Injectable silk fibroin foams and uses thereof

ABSTRACT

The inventions provided herein relate to compositions, methods, delivery devices and kits for repairing or augmenting a tissue in a subject. The compositions described herein can be injectable such that they can be placed in a tissue to be treated with a minimally-invasive procedure (e.g., by injection). In some embodiments, the composition described herein comprises a compressed silk fibroin matrix, which can expand upon injection into the tissue and retain its original expanded volume within the tissue for a period of time. The compositions can be used as a filler to replace a tissue void, e.g., for tissue repair and/or augmentation, or as a scaffold to support tissue regeneration and/or reconstruction. In some embodiments, the compositions described herein can be used for soft tissue repair or augmentation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application under 35 U.S.C.§371 of international PCT application no. PCT/US2012/064471, filed Nov.9, 2012, which claims the benefit of and priority to U.S. ProvisionalApplication No. 61/557,610, filed Nov. 9, 2011. The entire content ofthese applications are hereby incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. EB002520awarded by the National Institutes of Health and W81XWH-08-2-0032awarded by the US Army. The government has certain rights in theinvention.

TECHNICAL FIELD OF THE DISCLOSURE

The inventions described herein generally relate to silk fibroin-basedmaterials for biomedical applications, e.g., in soft tissue repair,augmentation and/or reconstruction.

BACKGROUND

The restoration of soft tissue defects from trauma, surgical excision orcongenital defects should start with a strategy that will maintaintissue size and shape to near normal dimensions for extended timeframes. Current clinical strategies include free fat transfers andartificial fillers. In the case of breast cancer patients receivingmastectomies, silicone shells filled with saline or silicone are used toreplace the void. This leaves the patient with an unnatural look andfeel, and the risk of capsular contracture resulting in a revisionsurgery. The fat grafting and artificial filler options fail to retainvolume over time. Thus, the fat grafting and artificial filler optionscan require a second surgical site, have avascular necrosis andgenerally do not regenerate the original tissue.

Bovine and human collagen have gained widespread use as injectablematerials for soft tissue augmentation and filling. Collagen, theprincipal extracellular structural protein of the animal body, has beenused as an implant material to replace or augment connective tissue,such as skin, tendon, cartilage and bone. Additionally, collagen hasbeen injected or implanted into the human body for cosmetic purposes fora number of years. However, the use of collagen in soft tissueaugmentation and/or filling could be costly and it does not have a longlasting effect, e.g., the results often only last for about 3 months.

Hyaluronic acid (HA) is a glycosaminoglycan that is naturally found inthe human body and is widely distributed throughout connective,epithelial, and neural tissues. Compositions of non-crosslinkedhyaluronic acid tend to degrade within a few months after injection andthus require fairly frequent reinjection to maintain their soft tissueaugmenting effect. More recently, compositions of cross-linkedhyaluronic acid have been used for soft tissue augmentation. However,such cross-linked compositions contain fairly large particles, aroundapproximately 2 mm each, of hyaluronic acid suspended in a gel. Whilethe larger particles could have a longer lasting effect, the largerparticle size can make the injection more challenging and create anunpleasant experience to a recipient.

In summary, the major disadvantages of the current strategies for softtissue regeneration, repair and/or augmentation include a large amountof tissues required for grafting large tissue defects; donor sitemorbidity, possibility of second surgical site, avascular necrosis; lossof shape and/or size of the scaffolds over time; material mismatch withnative tissue; and failure to regenerate tissue. Accordingly, there is astrong need to develop a strategy or a scaffold that can be administeredwith a minimally invasive procedure and will provide sustained retentionof volume restoration for at least 3 months or longer, e.g., for atleast 6 months or at least one year, while the body gradually remodelsand regenerates the site into near-normal tissue structure and function.

SUMMARY

Spongy biomaterial scaffolds such as foams are desirable in tissueengineering, e.g., for soft tissue regeneration, repair and/oraugmentation, partly because the network of interconnected pores withinthe spongy scaffolds is advantageous for cell attachment, yet allowingnutrient and waste flows. However, the mechanical property and/orstructure of the current biomaterial foams generally fail to regeneratetissue or retain their volume within the tissue for an extended periodof time. In addition, placement of such spongy biomaterial scaffolds inthe tissue can be invasive. Therefore, it is imperative to develop aminimally-invasive strategy for repairing or augmenting a tissue in anindividual, e.g., developing an injectable foam where the injectablefoam can retain their volume and shape while the tissue graduallyregenerates to restore its structure and function.

Embodiments of various aspects described herein are based on, at leastin part, our discovery that the silk fibroin-based foam can be injectedto fill void space in soft tissue, for example, soft tissue lost due toinjury or disease—such as in trauma, cancer resection surgeries, breastreconstruction, breast augmentation, and related needs. In addition, thesilk fibroin-based foam constructs can also be used for cosmeticpurposes, for example, as a more cost-effective and natural alternativeto topical treatments or BOTOX® treatments.

Accordingly, one aspect provided herein is an injectable composition foruse in repairing or augmenting a tissue in a subject, comprising acompressed silk fibroin matrix, wherein the compressed silk fibroinmatrix expands upon injection into the tissue, and retains at least aportion (e.g., at least about 1%, at least about 5%, at least about 10%,at least about 25%, at least about 50% or more) of its original expandedvolume within the tissue to be repaired or augmented for a period oftime (e.g., at least about 2 weeks, at least about 3 weeks, at leastabout 4 weeks, at least about 5 weeks, at least about 6 weeks orlonger).

Another aspect provided herein relates to a method for repairing oraugmenting a tissue in a subject. The method includes placing in thetissue to be repaired or augmented a composition comprising a compressedsilk fibroin matrix, wherein the compressed silk fibroin matrix expandsupon placement into the tissue, and retains at least a portion of itsoriginal expanded volume (e.g., at least about 1%, at least about 5%, atleast about 10%, at least about 25%, at least about 50% or more) withinthe tissue for a period of time (e.g., at least about 2 weeks, at leastabout 3 weeks, at least about 4 weeks, at least about 5 weeks, at leastabout 6 weeks or longer). In one embodiment, the composition is placedinto the tissue to be repaired or augmented by injection.

In some embodiments, the silk fibroin matrix can be provided in acompressed state for the treatment methods described herein. In someembodiments, the silk fibroin matrix can be provided in an uncompressedstate, and can then be compressed to a smaller volume during loadinginto a delivery applicator (e.g., an injection applicator such as aneedle, cannula, and/or a catheter) before placing into a tissue to berepaired or augmented.

In some embodiments of the compositions and methods provided herein, thecompressed silk fibroin matrix after placed (e.g., injected) into thetissue can expand in volume by at least about 50%, at least about 100%,at least about 2-fold, at least about 3-fold or more, as compared to thecompressed volume of the silk fibroin matrix.

In certain embodiments of the compositions and methods provided herein,the silk fibroin matrix can exclude an amphiphilic peptide. In otherembodiments, the silk fibroin matrices can include an amphiphilicpeptide. An exemplary amphiphilic peptide, for example, can comprise aRGD motif.

In some embodiments of the compositions and methods provided herein, thesilk fibroin matrix can retain at least about 50% of its expandedoriginal volume, including at least about 60%, at least about 70%, atleast about 80% or more, of its original expanded volume within thetissue for a period of time.

In some embodiments of the composition and method provided herein, thesilk fibroin matrix can retain at least a portion of its originalexpanded volume for at least about 6 weeks, at least about 3 months, atleast about 6 months or longer.

Volume retention of the silk fibroin matrix can be, in part, controlledby modulating the degradation and/or solubility properties of the silkfibroin matrix. For example, the silk fibroin matrix can be adapted todegrade at a pre-determined rate such that the silk fibroin matrix canmaintain a desirable volume over a pre-determined period of time, e.g.,to promote tissue regeneration or repair. For example, in suchembodiments, the silk fibroin matrix can be adapted to degrade no morethan 50% of its original expanded volume, for example, including no morethan 30%, no more than 10%, of its original expanded volume, in at leastabout 2 weeks, including at least about 6 weeks, at least about 3months, at least about 6 months or longer.

In some embodiments, the silk fibroin matrix can be adapted to degradeat a pre-determined rate such that the volume of the silk fibroin matrixgradually decreases (while still providing sufficient support) as atissue placed with the silk fibroin matrix begins to regenerate. In suchembodiments, the silk fibroin matrix can be adapted to degrade at leastabout 5% of its original expanded volume, for example, including atleast about 10%, at least about 20%, at least about 30% or more, of itsoriginal expanded volume, in at least about 2 weeks, including at leastabout 6 weeks, at least about 3 months, at least about 6 months orlonger.

Depending on the defect size of the tissue and/or desired properties ofthe silk fibroin matrix, the silk fibroin matrix (prior to compression)can be adapted to be any size. In some embodiments, the silk fibroinmatrix (prior to compression) can have a size of about 1 mm to about 5mm in diameter. In some embodiments, the silk fibroin matrix (prior tocompression) can have a size larger than 5 mm in diameter. Since thesilk fibroin matrix is compressible, the size of the silk fibroin matrixcan be as large as feasible to fill larger sized defects provided thatthe size of the compressed silk fibroin matrix is feasible for injectioninto a tissue.

The silk fibroin matrix can be adapted to mimic the structuralmorphology of native tissues and/or to deliver an active agent to alocal area of a tissue. For example, the silk fibroin matrix can beporous. In some embodiments, the porosity of the silk fibroin matrix canbe adapted to mimic the structural morphology and/or gradient ofcellular densities found in native tissue. In some embodiments, theporosity of the silk fibroin matrix can be adapted to deliver an activeagent to a tissue in a pre-determined release profile. In someembodiments, the porosity of the silk fibroin matrix can be adapted toretain at least a portion of its original expanded volume for a periodof time. For example, the silk fibroin porous matrix can have a porosityof at least about 1%, including, e.g., at least about 3%, at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 70%, at least about 80%, at least about 90% or higher.The pore size of such porous silk fibroin matrix can range from about 1μm to about 1500 μm, from about 50 μm to about 650 μm, or from about 100μm to about 600 μm.

The silk fibroin matrix, in one embodiment, can be fabricated byfreeze-processing a silk fibroin solution. In some embodiments, the silkfibroin solution can have a concentration of about 0.5% w/v to about 10%w/v, or about 1% w/v to about 6% w/v.

In some embodiments of any aspects described herein, the silk fibroinmatrix can be a silk fibroin foam.

In some embodiments, the silk fibroin matrix or the compositionsdescribed herein can further comprise a hydrogel material.

The injectable composition described herein comprising the silk fibroinmatrix can further comprise at least one active agent. In someembodiments, the silk fibroin matrix of the composition described hereincan further comprise at least one active agent. Non-limiting examples ofthe active agents can include biologically active agents, cosmeticallyactive agents, cell attachment agents, a dermal filler material, and anycombinations thereof. In some embodiments, the active agent can be acell, e.g., without limitations, a stem cell. In some embodiments, theactive agent can be an adipose-derived stem cell. In some embodiments,the active agent can be a biological fluid or concentration, e.g.,without limitations, a lipoaspirate or a bone marrow aspirate. In someembodiments, the active agent can be a therapeutic agent. In someembodiments, the active agent can be a cosmetically active agent. Insome embodiments, the active agent can be a dermal filler material.

Various embodiments of the composition described herein can be injectedinto a tissue to be repaired or augmented by any known methods in theart, e.g., subcutaneously, submuscularly, or intramuscularly. Wheninjected in a tissue, some embodiments of the composition can be atleast partially dry. Alternatively, the composition can be at leastpartially hydrated. In some embodiments, the composition can furthercomprise a carrier, e.g., a buffered solution and/or a biological fluidor concentrate (e.g., a lipoaspirate), when injected in a tissue.

The tissue to be repaired or augmented by the composition and/or themethod described herein can be a soft tissue. Exemplary examples of asoft tissue include, but are not limited to, a tendon, a ligament, skin,a breast tissue, a fibrous tissue, a connective tissue, a muscle, andany combinations thereof. In certain embodiments, the soft tissue isskin. In other embodiments, the soft tissue is a breast tissue.

A delivery device comprising one embodiment of an injectable compositionand/or silk fibroin matrix is also provided herein. A delivery devicecan include an injection device (e.g., in a form of a syringe or aninjection gun) and/or any administration device that is minimallyinvasive. Accordingly, in some embodiments, provided herein relate to aninjection device comprising an injectable composition described herein.The delivery or injection device can further comprise a tubularstructure for introducing the silk fibroin matrix or the compositiondescribed herein into a tissue to be repaired or augmented. The tubularstructure can be tapered (e.g., comprising a conical interior space),e.g., to facilitate loading of the compressed silk fibroin matrixtherein. In some embodiments, the tubular structure can permitcompression of a silk fibroin matrix to a pre-determined volume (e.g.,interior volume of the tubular structure) while loading the silk fibroinmatrix therein. Examples of the tubular structure include, but are notlimited to, a needle, a cannula, a catheter, or any combinationsthereof. In some embodiments, the tubular structure can be pre-loadedwith the silk fibroin matrix. In this embodiment, the silk fibroinmatrix can be in a compression state inside the tubular structure. Insome embodiments, the delivery or injection device can further comprisea mechanical element (e.g., an elongated rod-like structure) tofacilitate the exit of the compressed silk fibroin matrix through thetubular structure. In some embodiments, the delivery or injection devicecan further comprise an injection carrier. In some embodiments, thedelivery or injection device can further comprise a local anesthetic.

In some embodiments of any aspects described herein, the compositionsand/or delivery devices can be stored or transported at a temperatureabout 0° C. and about 60° C., e.g., between about 10° C. and about 60°C. or between about 15° C. and about 60° C. At such temperatures, thebioactivity of active agents embedded or distributed inside the silkfibroin matrix can be stabilized for a period of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show images of silk fibroin-based injectable foam disks inaccordance with one or more embodiments described herein. FIG. 1A showsan image of a silk fibroin-based injectable foam disk being excised,e.g., using a 4 mm biopsy punch. FIG. 1B shows an image of a silkfibroin-based injectable foam disk after excision.

FIGS. 2A-2B show images of an exemplary method of placing a silkfibroin-based injectable foam disk into an injectable position inside aninjector tip (e.g., a pipette tip). FIG. 2A shows an image of a silkfibroin-based injectable foam disk being loaded into a pipette tip. FIG.2B shows an image of a silk fibroin-based injectable foam disk beingtamped into an injection position inside a pipette tip, e.g., using astiff wire.

FIGS. 3A-3D show images of an exemplary method of injecting a silkfibroin-based injectable foam disk into a tissue. FIG. 3A shows an imageof puncturing a hole in the tissue of the chicken thigh, e.g., using astraight 14-gauge needle. FIG. 3B shows an image of inserting into ahole a pipette tip containing a silk fibroin-based injectable foam disk(e.g., as shown in FIG. 2B). FIG. 3C shows an image of ejecting the silkfibroin-based foam disk, e.g., using a stiff wire, into the tissue whileslowly drawing out the pipette tip. FIG. 3D shows an image of theinjectable silk fibroin-based foam disk positioned in the raw chickenthigh (black arrow denotes the silk fibroin-based foam disk injectedinto the tissue).

FIGS. 4A-4F show images of an exemplary method of extracting an injectedsilk fibroin-based foam from a tissue. FIG. 4A shows an image ofpalpation of a tissue (e.g., raw chicken tissue) for the injected silkfibroin-based foam. FIG. 4B shows an image of an incision (e.g., madewith a razor blade) close to where the embedded silk fibroin-based foamis located. FIG. 4C shows an image of exposure of the embedded silkfibroin-based foam after the incision. FIG. 4D shows another perspectiveview of the silk fibroin-based foam from FIG. 4C in cross-section. FIG.4E shows an image of removing the exposed silk fibroin-based foam (e.g.,using a pair of tweezers). FIG. 4F shows an image of the silkfibroin-based foam extracted from the tissue.

FIG. 5 shows an exemplary method of using one or more embodiments of theinjectable compositions described herein. The porous silk fibroin-basedfoams (e.g., formed by freezer processing) can be mixed withlipoaspirate as a carrier, optionally containing adipose-derived stemcells (ASCs), to form an exemplary injectable composition. Theinjectable compositions can then be injected into a subject, e.g., ananimal model.

FIGS. 6A-6C show images of another exemplary method of injecting a silkfibroin-based injectable foam disk into a tissue-like material. FIG. 6Ais a series of images showing steps of preparing a catheter forinjecting a silk fibroin-based foam into a tissue. A hole is puncturedin a target area of the tissue, so that a catheter can be inserted intothe hole. FIG. 6B is a series of images showing steps of loading a silkfibroin-based foam into the catheter inserted into the tissue. Afterinserting the catheter into a tissue, a silk fibroin-based foam isloaded into the catheter, e.g., via an adaptor. FIG. 6C is a series ofimages showing steps of pushing a silk fibroin-based foam through acatheter. After loading the silk fibroin-based foam into the adaptor tothe catheter, a rod is used to push the foam down through the catheterinto the tissue.

FIGS. 7A-7B show images of an exemplary method of injecting a silkfibroin-based foam into a tissue in vivo. FIG. 7A is an exemplarycustom-modified injection gun for use to facilitate the injection of asilk fibroin-based foam into a tissue in vivo. A foam ramrod is beingplaced into the injection gun. FIG. 7B is a series of images showingexemplary steps of injecting a silk fibroin-based foam into a tissue ina rat or mouse model. A catheter (e.g., with a gauge of 14 G) isinserted into a target tissue area, followed by a silk fibroin-basedfoam loaded into the catheter. The catheter is then connected to a foaminjector (e.g., the injection gun as shown in FIG. 7A), so that the silkfibroin-based foam can be injected slowly through the catheter into thetarget tissue area (e.g., subcutaneous area).

FIGS. 8A-8G show images and results of some embodiments of the silkfibroin-based foams injected into a rat model in vivo. FIGS. 8A-8C showimages of silk fibroin-based foams injected into a rat model in vivoafter the removal of the rat skin. Silk fibroin-based foams producedfrom different concentrations of silk fibroin solution (e.g., 1%, 3%, 6%silk fibroin) and sources of cocoon (Japanese: JP vs. Taiwanese: TW)were evaluated after injection for 1 day (FIG. 8A), 14 days (FIG. 8B)and 30 days (FIG. 8C). FIG. 8A shows that the injected silkfibroin-based foams remained clear 1 day after injection, unless theywere stained by blood due to a puncture into a blood vessel (e.g., TW3).FIGS. 8B-8C show that the injected silk fibroin-based foams obtained areddish hue about 14 days and about 30 days, respectively, afterinjection. However, there appeared no significant change invascularization leading to the injected foams. FIG. 8D shows an image ofthe injected foams visible from outside skin of a rat. FIG. 8E is a setof images showing gross morphology of some embodiments of the silkfibroin-based injectable foams (corresponding to the ones in FIGS.8A-8C) explanted after an indicated post-injection period (e.g., 1 day,14 days and 30 days post-injection). There are no observable visualdifferences in gross morphology at the indicated timepoints. The silkfibroin foams are consistently stiffer with increased silk weightpercentage. All explants are soft to the touch and return to theiroriginal shape after deformation. FIG. 8F shows the volume retentionresults of the silk fibroin-based foams after injection into the tissuefor 1 day or 14 days. FIG. 8G shows the volume retention results of thesilk fibroin-based foams after injection into the tissue for 14 days, 30days or 60 days. The results of FIGS. 8F and 8G are expressed inpercents of volume retained relative to the original volume.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods, compositions, delivery devices, and kitsfor repairing or augmenting a tissue in a subject. In accordance withembodiments of various aspects described herein, a reversibly-deformableand/or injectable format of silk fibroin scaffolds (e.g., silk fibroinfoams) can be compressed (e.g., to a smaller volume) and placed (e.g.,by injection) into a tissue to be repaired or augmented. Upon placement(e.g., injection) into the tissue, the compressed silk fibroin matrixexpands to a volume (e.g., an increase in volume by at least about 10%of its compressed volume) and retains at least a portion of its originalexpanded volume (e.g., at least about 50% of its original expandedvolume or more) within the tissue to be repaired or augmented for aperiod of time (e.g., at least about 2 weeks or longer). Suchreversibly-deformable and/or injectable silk fibroin matrix can beintroduced into a defect site with a minimally-invasive procedure.

Silk Fibroin Matrix

Silk fibroin matrices described herein are deformable. The silk fibroinmatrices can be compressed prior to and/or during placement (e.g.,injection) into a tissue to be repaired or augmented. Upon placement(e.g., injection) into the tissue, the compressed silk fibroin matricescan then expand within the tissue and retain its original expandedvolume within the tissue for a period of time.

As used herein, the term “deformable” generally refers to the ability ofan object to change size (e.g., volume) and/or shape in response to anexternal pressure/force, while maintaining the integrity of the object(i.e., the object remains intact as a whole, without breaking intopieces, during deformation, and has the ability to restore at least aportion of its original size and shape). With respect to a deformablesilk fibroin matrix, the silk fibroin matrix can decrease its volumeand/or change its shape when compressed by an applied force such thatthe silk fibroin matrix becomes small enough (but intact) to be loadedinto an injection applicator (e.g., a needle, a cannula, or a catheter)having a dimension much smaller than that of the uncompressed silkfibroin matrix, and/or that the silk fibroin matrix are compressed toadopt the cross-sectional shape of the injection applicator.

As used herein, the term “compressed” generally refers to a decrease involume of a silk fibroin matrix. In some embodiments, a decrease involume of a silk fibroin matrix can also lead to a change in one or morephysical properties of the silk fibroin matrix, e.g., an increase inoriginal density (before compression) of the silk fibroin matrix, adecrease in original pore size (before compression) and/or originalporosity (before compression) of the silk fibroin matrix. Compression ofa silk fibroin matrix to a pre-determined volume can be performed by anyknown methods in the art, e.g., by physical loading of a silk fibroinmatrix into the interior space of a delivery applicator (e.g., a needle,cannula or a catheter), or by vacuum.

In some embodiments, the silk fibroin matrix can be compressed to avolume of no more than 80% of its original volume (i.e., the volume ofthe silk fibroin matrix before compression), including no more than 70%,no more than 60%, no more than 50%, no more than 40%, no more than 30%,no more than 20%, no more than 10%, no more than 5% or lower, of itsoriginal volume (i.e., the volume of the silk fibroin matrix beforecompression). In some embodiments, the silk fibroin matrix can becompressed to a volume of at least about 10% of its original volume(i.e., the volume of the silk fibroin matrix before compression),including at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90% or more, but excluding 100%, of its originalvolume (i.e., the volume of the silk fibroin matrix before compression).In some embodiments, silk fibroin matrix, such as silk fibroin foams,fabricated from a silk fibroin solution with concentrations of about 1%to about 6% can be compressed to approximately 20% to 30% of theiroriginal volume. The amount of compression possible without breaking orcausing permanent deformation of the silk fibroin matrix is dependenton, for example, the material properties, silk fibroin concentration,and/or fabrication/process methods and parameters. In some embodiments,higher molecular weight of the matrix or other improved processing canyield higher levels of compression of a silk fibroin-based matrix, e.g.,a silk fibroin-based matrix compressed to less than 20% of its originalvolume.

In some embodiments, the silk fibroin matrix can be compressed to avolume that increase the original density (e.g., ratio of weight touncompressed volume) of the silk fibroin matrix by at least about 10%,including, e.g., at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about1-fold, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, or more.

After the compressed silk fibroin matrix is released from a deliveryapplicator (e.g., an injection applicator) and is placed (e.g.,injected) into a tissue, the silk fibroin matrix can expand in responseto the removal of the compressive stress, the size of a void in thetissue, the mechanical properties of the tissue and the silk fibroinmatrix, and any combinations thereof. In some embodiments, thecompressed silk fibroin matrix can expand and restore the originalvolume of the silk fibroin matrix (i.e., the volume of the silk fibroinmatrix before compression). In some embodiments, the compressed silkfibroin matrix can expand to a size sufficient to fill a void in thetissue to be repaired or augmented. In some embodiments, the silkfibroin matrix can expand to a size such that the silk fibroin matrixand the tissue surrounding the silk fibroin matrix are pressing eachother with an equilibrium force. In certain embodiments, the compressedsilk fibroin matrix can expand, upon placement (e.g., injection) into atissue to be repaired or augmented, to a size in volume of at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, or up to andincluding 100% of the original volume (i.e., the volume of the silkfibroin matrix before compression). In some embodiments, the compressedsilk fibroin matrix can expand in volume upon placement (e.g.,injection) into a tissue to be repaired or augmented, by at least about1-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 6-fold or higher, as compared to the compressedvolume (e.g., the volume of the silk fibroin matrix being compressedinside a delivery applicator, e.g., an injection applicator, e.g., aneedle, a cannula or a catheter). In such embodiments, without wishingto be bound by theory, the silk fibroin matrix can expand beyond itsoriginal volume, partly because the silk fibroin matrix can absorbmoisture or water from the surrounding tissue, causing it to swell.

In some embodiments, stated another way, the compressed silk fibroinmatrix can expand in volume by at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at least aabout 80%, at least about 90%, at least about 95%, at least about1-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 6-fold, at least about 7-fold, at least about8-fold, at least about 9-fold, at least about 10-fold, or more, relativeto the volume of the compressed silk fibroin matrix.

The expansion of the silk fibroin matrix within the tissue to reach aplateau volume can be spontaneous (e.g., within 5 seconds or less) oroccur over a period of time, e.g., seconds, minutes, and hours. In someembodiments, at least about 50% of the increase in volume of the silkfibroin matrix can occur in less than 3 hours, less than 2 hours, lessthan 1 hour, less than 30 mins, less than 20 mins, less than 10 mins,less than 5 minutes or shorter, upon injection of the silk fibroinmatrix into the tissue, while the remaining increase in volume of thesilk fibroin matrix can occur over a much longer time scale. Theexpansion rate of the silk fibroin matrix within the tissue can dependon several factors, including, but not limited to, hydration state,pressure, and volume of void space, as well as silk material properties,foam structural properties (including porosity), and interaction betweenthe fluid and structure. For example, if the silk fibroin matrix (e.g.,silk fibroin foam) is injected into a fluid-filled void, the expansioncan be rapid. If a dry silk fibroin matrix (e.g., a dry silk fibroinfoam) is injected, much slower hydration and expansion is likely tooccur, e.g., from minutes to an hour.

After the silk fibroin matrix expands upon injection into the tissue,the silk fibroin matrix can retain at least a portion (e.g., at leastabout 50% or more) of its original expanded volume within the tissue forat least a period of time (e.g., at least about 2 weeks, at least about4 weeks, at least about 6 weeks or longer).

By “original expanded volume” in reference to the silk fibroin matrixdescribed herein is generally meant the volume of the silk fibroinmatrix as measured after it has expanded upon injection within a tissueto be repaired or augmented, or the corresponding increase in tissuevolume as measured after the silk fibroin matrix has expanded uponinjection. For example, the original expanded volume of the silk fibroinmatrix can be measured, for example, as soon as there is no significantincrease in the volume of the silk fibroin matrix for at least about 72hours, at least about 48 hours, at least about 24 hours, at least about12 hours, at least about 6 hours, at least about 3 hours or less, uponinjection of the silk fibroin matrix or the injectable composition intothe tissue. Stated another way, the original expanded volume of the silkfibroin matrix can be determined by measuring the corresponding increasein tissue volume (due to the expansion of the silk fibroin matrix), assoon as there is no significant increase in the tissue volume for atleast about 72 hours, at least about 48 hours, at least about 24 hours,at least about 12 hours, at least about 6 hours, at least about 3 hoursor less, upon injection of the silk fibroin matrix or the injectablecomposition into the tissue. In some embodiments, the original expandedvolume can refer to the original volume of the silk fibroin matrix(i.e., the volume of the silk fibroin matrix before compression).

As used herein, the term “retain” refers to maintaining the volume(e.g., size and/or shape) of the silk fibroin matrix described hereinover a period of time. In some embodiments, the silk fibroin matrix canretain over a period of time at least about 20% of its original expandedvolume, including, for example, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90% of its original expanded volume or higher.In some embodiments, the silk fibroin matrix can retain over a period oftime at least about 1% of its original expanded volume, including, forexample, at least about 3%, at least about 5%, at least about 10%, atleast about 15%, at least about 20% of its original expanded volume orhigher. In some embodiments, the silk fibroin matrices can retain 100%of its original expanded volume, e.g., no detectable changes in thevolume, within the tissue to be repaired or augmented for a period oftime. In one embodiment, the silk fibroin matrix can retain at leastabout 1% of its original expanded volume within the tissue to berepaired or augmented for a period of time. In one embodiment, the silkfibroin matrix can retain at least about 50% of its original expandedvolume within the tissue to be repaired or augmented for a period oftime. In one embodiment, the silk fibroin matrix can retain at leastabout 60% of its original expanded volume within the tissue to berepaired or augmented for a period of time. In one embodiment, the silkfibroin matrix can retain at least about 70% of its original expandedvolume within the tissue to be repaired or augmented for a period oftime. In one embodiment, the silk fibroin matrix can retain at leastabout 80% of its original expanded volume within the tissue to berepaired or augmented for a period of time. The volume of the silkfibroin matrix placed into a tissue can be determined or indicated by achange in at least one of the tissue properties, e.g., tissue volume,tissue elasticity, and/or tissue hardness. In some embodiments, thevolume of the silk fibroin matrix placed into a tissue can be determinedfrom explants.

The silk fibroin matrix can retain at least a portion of its originalexpanded volume for any period of time, e.g., weeks, months, or years.In some embodiments, the silk fibroin matrix can retain, e.g., at leastabout 1% of its original expanded volume (including e.g., at least about3%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, or higher,of their original volume) for at least about 2 weeks, at least about 3weeks, at least about 4 weeks, at least about 5 weeks, at least about 6weeks, at least about 7 weeks, at least about 8 weeks, at least about 3months, at least about 4 months, at least about 5 months, at least about6 months, at least about 7 months, at least about 8 months, at leastabout 9 months, at least about 10 months, at least about 11 months, atleast about 1 year, at least about 2 years, at least 3 years, at leastabout 4 years, at least 5 years or longer. In certain embodiments, thesilk fibroin matrix can retain, e.g., at least about 70% of its originalexpanded volume or higher, for at least about 3 months or longer. Inother embodiments, there can be no significant changes in the volume ofthe silk fibroin matrix or the corresponding increase in tissue volumeafter placed into a tissue to be repaired or augmented for at leastabout 3 months or longer. In some embodiments, the silk fibroin matrixcan retain, e.g., at least about 70% of its original expanded volume orhigher, for at least about 6 months or longer (including, e.g., at leastabout 9 months, at least about 12 months, at least about 18 months orlonger). In other embodiments, there can be no significant changes inthe volume of the silk fibroin matrix or the corresponding increase intissue volume after placed into a tissue to be repaired or augmented forat least about 6 months or longer. In particular embodiments, the silkfibroin matrix can retain at least about 20% of its original expandedvolume or higher for at least about 1 year or longer (including, e.g.,at least about 2 years, at least about 3 years, at least about 4 years,at least about 5 years or longer). In some embodiments, the silk fibroinmatrix can retain at least about 50% of its original expanded volume orhigher for at least about 1 year or longer (including, e.g., at leastabout 2 years, at least about 3 years, at least about 4 years, at leastabout 5 years or longer). In some embodiments, the silk fibroin matrixcan retain at least about 1% of its original expanded volume or higherfor at least about 1 year or longer (including, e.g., at least about 2years, at least about 3 years, at least about 4 years, at least about 5years or longer).

The volume retention of the silk fibroin matrix can also becharacterized by, e.g., degradation of the silk fibroin matrix.Generally, the slower the silk fibroin matrix degrades, the longer thesilk fibroin matrix can retain its original expanded volume in a tissue.Accordingly, some embodiments provided herein are directed to injectablecompositions for use in repairing or augmenting a tissue in a subject,the compositions comprising a compressed silk fibroin matrix, whereinthe compressed silk fibroin matrix expands upon injection into thetissue, and the silk fibroin matrix is adapted to degrade within thetissue to be repaired or augmented over a period of time.

As used in reference to the silk fibroin matrix described herein, theterm “degrade” or “degradation” refers to a decrease in volume or sizeof the silk fibroin matrix. The degradation of the silk fibroin matrixcan occur via cleavage of the silk fibroin matrix into smaller fragmentsand/or dissolution of the silk fibroin matrix or fragments thereof. Insome embodiments, the silk fibroin matrix can be adapted to degrade nomore than 80% of its original expanded volume, including, for example,no more than 70%, no more than 60%, no more than 50%, no more than 40%,no more than 30%, no more than 20%, no more than 10% of its originalexpanded volume or lower. In some embodiments, the silk fibroin matrixcan exhibit no significant degradation (e.g., no detectable changes inthe volume) within the tissue to be repaired or augmented. In oneembodiment, the silk fibroin matrix can be adapted to degrade no morethan 50% of its original expanded volume within the tissue to berepaired or augmented for a period of time. In one embodiment, the silkfibroin matrix can be adapted to degrade no more than 40% of itsoriginal expanded volume within the tissue to be repaired or augmentedfor a period of time. In one embodiment, the silk fibroin matrix can beadapted to degrade no more than 30% of its original expanded volumewithin the tissue to be repaired or augmented for a period of time. Inone embodiment, the silk fibroin matrix can be adapted to degrade nomore than 20% of its original expanded volume within the tissue to berepaired or augmented for a period of time. In one embodiment, the silkfibroin matrix can be adapted to degrade no more than 10% of itsoriginal expanded volume within the tissue to be repaired or augmentedfor a period of time.

In some embodiments, the silk fibroin matrix can be adapted to degradeat a pre-determined rate such that the original expanded volume of thesilk fibroin matrix gradually decreases (while still providingsufficient support) as a tissue placed with the silk fibroin matrixbegins to regenerate. In such embodiments, the silk fibroin matrix canbe adapted to degrade at least about 5% of its original expanded volume,for example, including at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95% or more, of its original expanded volume over a pre-determinedperiod of time (e.g., a period of at least about 2 weeks, including atleast about 6 weeks, at least about 3 months, at least about 6 months orlonger.)

The silk fibroin matrix can be adapted to degrade at any rate. In someembodiments, the silk fibroin matrix can be adapted to degrade at leasta portion of its original expanded volume over any period of time, e.g.,weeks, months, or years. In some embodiments, the silk fibroin matrixcan be adapted to degrade at least a portion of its original expandedvolume, e.g., no more than 50% of its original expanded volume(including e.g., no more than 40%, no more than 30%, no more than 20% orlower, of its original expanded volume), in at least about 2 weeks, atleast about 3 weeks, at least about 4 weeks, at least about 5 weeks, atleast about 6 weeks, at least about 7 weeks, at least about 8 weeks, atleast about 3 months, at least about 4 months, at least about 5 months,at least about 6 months, at least about 7 months, at least about 8months, at least about 9 months, at least about 10 months, at leastabout 11 months, at least about 1 year, at least about 2 years, at leastabout 3 years, at least about 4 years, at least about 5 years or longer.In certain embodiments, the silk fibroin matrix can be adapted todegrade, e.g., no more than 30% of its original expanded volume orlower, in at least about 3 months or longer. In other embodiments, therecan be no significant degradation (i.e., no detectable changes in thevolume of the silk fibroin matrix) after placed into a tissue to berepaired or augmented for at least about 3 months or longer. In someembodiments, the silk fibroin matrix can be adapted to degrade, e.g., nomore than 30% of its original expanded volume or lower, in at leastabout 6 months or longer (including, e.g., at least about 9 months, atleast about 12 months, at least about 18 months or longer). In otherembodiments, there can be no significant degradation (i.e., nodetectable changes in the volume of the silk fibroin matrix) afterplaced into a tissue to be repaired or augmented for at least about 6months or longer. In particular embodiments, the silk fibroin matrix canbe adapted to degrade no more than 80% of its original expanded volumeor lower in at least about 1 year or longer (including, for example, atleast about 2 years, at least about 3 years, at least about 4 years, atleast about 5 years or longer). In some embodiments, the silk fibroinmatrix can be adapted to degrade no more than 50% of its originalexpanded volume or lower in at least about 1 year or longer.

The same or similar formulation of the silk fibroin matrix or injectablecompositions can manifest different responses in a subject. By way ofexample only, the volume retention or degradation rate of the silkfibroin matrix in a tissue can vary from one subject to another, e.g.,because of different tissue microenvironment such as species and/orlevels of various proteins or enzymes (e.g., proteolytic enzymes)present in the tissue.

In some embodiments, the silk fibroin matrix can be adapted to maintaina constant volume retention rate and/or degradation rate over a periodof time. In some embodiments, the silk fibroin matrix can be adapted tohave a volume retention rate or degradation rate varying with time. Forexample, the silk fibroin matrix can be coated with a polymeric materialor a biomaterial, e.g., silk fibroin of a different concentration and/ora different biodegradable and biocompatible polymer. Such coating canpossess a different function and/or a different degradation rate fromthat of the silk fibroin matrix core. By way of example only, thecoating of the silk fibroin matrix can contain at least one active agentand be adapted to degrade at a different rate (e.g., at a faster rate)from that of the silk fibroin matrix core. Thus, upon placing the silkfibroin matrix in a tissue, the coating of the silk fibroin matrix canbe adapted to degrade faster, e.g., to release the active agent forrelieving the pain and/or promoting the wound healing, while the core ofthe silk fibroin matrix can retain their volume for a longer period oftime.

Silk fibroin is a particularly appealing biopolymer candidate to be usedfor embodiments described herein, e.g., because of its versatileprocessing e.g., all-aqueous processing (Sofia et al., 54 J. Biomed.Mater. Res. 139 (2001); Perry et al., 20 Adv. Mater. 3070-72 (2008)),relatively easy functionalization (Murphy et al., 29 Biomat. 2829-38(2008)), and biocompatibility (Santin et al., 46 J. Biomed. Mater. Res.382-9 (1999)). For example, silk has been approved by U.S. Food and DrugAdministration as a tissue engineering scaffold in human implants. SeeAltman et al., 24 Biomaterials: 401 (2003).

As used herein, the term “silk fibroin” includes silkworm fibroin andinsect or spider silk protein. See e.g., Lucas et al., 13 Adv. ProteinChem. 107 (1958). Any type of silk fibroin can be used in differentembodiments described herein. Silk fibroin produced by silkworms, suchas Bombyx mori, is the most common and represents an earth-friendly,renewable resource. For instance, silk fibroin used in a silk film maybe attained by extracting sericin from the cocoons of B. mori. Organicsilkworm cocoons are also commercially available. There are manydifferent silks, however, including spider silk (e.g., obtained fromNephila clavipes), transgenic silks, genetically engineered silks, suchas silks from bacteria, yeast, mammalian cells, transgenic animals, ortransgenic plants (see, e.g., WO 97/08315; U.S. Pat. No. 5,245,012), andvariants thereof, that can be used. In some embodiments, silk fibroincan be derived from other sources such as spiders, other silkworms,bees, and bioengineered variants thereof.

In various embodiments, the silk fibroin can be modified for differentapplications and/or desired mechanical or chemical properties (e.g., tofacilitate formation of a gradient of active agent in silk fibroinmatrices). One of skill in the art can select appropriate methods tomodify silk fibroins, e.g., depending on the side groups of the silkfibroins, desired reactivity of the silk fibroin and/or desired chargedensity on the silk fibroin. In one embodiment, modification of silkfibroin can use the amino acid side chain chemistry, such as chemicalmodifications through covalent bonding, or modifications throughcharge-charge interaction. Exemplary chemical modification methodsinclude, but are not limited to, carbodiimide coupling reaction (see,e.g. U.S. Patent Application. No. US 2007/0212730), diazonium couplingreaction (see, e.g., U.S. Patent Application No. US 2009/0232963),avidin-biotin interaction (see, e.g., International Application No.: WO2011/011347) and pegylation with a chemically active or activatedderivatives of the PEG polymer (see, e.g., International Application No.WO 2010/057142). Silk fibroin can also be modified through genemodification to alter functionalities of the silk protein (see, e.g.,International Application No. WO 2011/006133). For instance, the silkfibroin can be genetically modified, which can provide for furthermodification of the silk such as the inclusion of a fusion polypeptidecomprising a fibrous protein domain and a mineralization domain, whichcan be used to form an organic-inorganic composite. See WO 2006/076711.Additionally, the silk fibroin matrix can be combined with a chemical,such as glycerol, that, e.g., affects flexibility of the matrix. See,e.g., WO 2010/042798, Modified Silk films Containing Glycerol.

As used interchangeably herein, the phrase “silk fibroin matrix” or“silk fibroin-based matrix” generally refer to a matrix comprising silkfibroin. In some embodiments, the phrases “silk fibroin matrix” and“silk fibroin-based matrix” refer to a matrix in which silk fibroinconstitutes at least about 30% of the total composition, including atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95% orhigher, of the total composition. In certain embodiments, the silkfibroin matrix or the silk fibroin-based matrix can be substantiallyformed from silk fibroin. In various embodiments, the silk fibroinmatrix or the silk fibroin-based matrix can be substantially formed fromsilk fibroin comprising at least one active agent. In some embodiments,the silk fibroin matrix or silk fibroin-based matrix can refer to a silkfibroin foam or a silk fibroin-based foam.

The silk fibroin matrix described herein can be adapted to be any shape,e.g., a spherical shape, polygonal-shaped, elliptical-shaped,cylindrical-shaped, tubular-shaped, or any art-recognized shapes. Thesize of the silk fibroin matrix can vary with a number of factorsincluding, without limitations, the size of the tissue to be repaired oraugmented and/or desired properties of the silk fibroin matrix, e.g.,volume retention or degradation profile. In some embodiments, the silkfibroin matrix (prior to compression) can have a size of about 1 mm toabout 5 mm in diameter. In some embodiments, the silk fibroin matrix(prior to compression) can have a size larger than 5 mm in diameter.Since the silk fibroin matrix are compressible or deformable, the sizeof the silk fibroin matrix can be as large as feasible to fill largersized defects as long as the size of the compressed silk fibroin matrixis feasible for injection into a tissue.

The silk fibroin matrices can be produced from aqueous-based or organicsolvent-based silk fibroin solutions. In some embodiments, the silkfibroin matrices produced from organic solvent-based silk fibroinsolution can retain their original volume for a longer period of timethan the aqueous-based silk fibroin matrices. The aqueous- or organicsolvent-based silk fibroin solution used for making silk fibroinmatrices described herein can be prepared using any techniques known inthe art. The concentration of silk fibroin in solutions used for softtissue repair or augmentation can be suited to the particular volumeretention requirement, e.g., if higher concentrations of silk fibroinsolutions can be used when longer volume retention of the silk fibroinmatrices is desired when injected into the tissue to be repaired oraugmented. In some embodiments, the silk fibroin solution for making thesilk fibroin matrices described herein can vary from about 0.1% (w/v) toabout 30% (w/v), inclusive. In some embodiments, the silk fibroinsolution can vary from about 0.5% (w/v) to about 10% (w/v). In someembodiments, the silk fibroin solution can vary from about 1% (w/v) toabout 6% (w/v). Suitable processes for preparing silk fibroin solutionare disclosed, for example, in U.S. Pat. No. 7,635,755; andInternational Application Nos: WO/2005/012606; and WO/2008/127401. Amicro-filtration step can be used herein. For example, the prepared silkfibroin solution can be processed further, e.g., by centrifugationand/or syringe based micro-filtration before further processing intosilk fibroin matrices described herein.

In some embodiments, the silk fibroin can be also mixed with otherbiocompatible and/or biodegradable polymers to form mixed polymermatrices comprising silk fibroin. One or more biocompatible and/orbiodegradable polymers (e.g., two or more biocompatible polymers) can beadded to the silk fibroin solution. The biocompatible polymer that canbe used herein include, but are not limited to, polyethylene oxide(PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin,polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronicacid, pectin, polycaprolactone, polylactic acid, polyglycolic acid,polyhydroxyalkanoates, dextrans, polyanhydrides, polymer, PLA-PGA,polyanhydride, polyorthoester, polycaprolactone, polyfumarate, collagen,chitosan, alginate, hyaluronic acid and other biocompatible and/orbiodegradable polymers. See, e.g., International Application Nos.: WO04/062697; WO 05/012606.

In some embodiments, at least one active agent described herein can beadded to the silk fibroin solution before further processing into silkfibroin matrices described herein. In some embodiments, the active agentcan be dispersed homogeneously or heterogeneously within the silkfibroin, dispersed in a gradient, e.g., using the carbodiimide-mediatedmodification method described in the U.S. Patent Application No. US2007/0212730.

In some embodiments, the silk fibroin matrices can be first formed andthen contacted with (e.g., dipped into) at least one active agent suchthat the open surface of the matrices can be coated with at least oneactive agent.

In some embodiments, the silk fibroin matrices described herein cancomprise porous structures, e.g., to mimic the structural morphology ofa native tissue, to modulate the degradation rate/volume retention rateof the silk fibroin matrices, and/or to modulate release profile of anactive agent embedded therein, if any. As used herein, the terms“porous” and “porosity” are generally used to describe a structurehaving a connected network of pores or void spaces (which can, forexample, be openings, interstitial spaces or other channels) throughoutits volume. The term “porosity” is a measure of void spaces in amaterial, and is a fraction of volume of voids over the total volume, asa percentage between 0 and 100% (or between 0 and 1).

In some embodiments, the porous silk fibroin matrices can be configuredto have any porosity, depending on the desired properties. For example,in some embodiments, the porous silk fibroin matrix can have a porosityof at least about 1%, at least about 3%, at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90% orhigher. In some embodiments, the porosity can range from about 70% toabout 99%, or from about 80% to about 98%. The pore size and totalporosity values can be quantified using conventional methods and modelsknown to those of skill in the art. For example, the pore size andporosity can be measured by standardized techniques, such as mercuryporosimetry and nitrogen adsorption. One of ordinary skill in the artcan determine the optimal porosity of the silk fibroin matrices forvarious purposes. For example, the porosity and/or pore size of the silkfibroin matrices can be optimized based on the desired degradation rateor volume retention rate of the silk fibroin matrices, release profilesof an active agent from the silk fibroin matrices, and/or the structuralmorphology of the tissue to be repaired or augmented.

The pores can be adapted to have any shape, e.g., circular, elliptical,or polygonal. The porous silk fibroin matrices can be adapted to have apore size of about 1 μm to about 1500 μm, about 10 μm to about 1000 μm,about 25 μm to about 800 μm, about 50 μm to about 650 μm, or about 100μm to about 600 μm. In some embodiments, the pores can have a size ofabout 100 μm to about 600 μm. In some embodiments, the silk fibroinmatrix can have a pore size of less than 1 μm. In other embodiments, thesilk fibroin matrix needs not be porous. In such embodiments, the poresize of the silk fibroin matrix can be less than 10 nm ornon-detectable. The term “pore size” as used herein refers to adimension of a pore. In some embodiments, the pore size can refer to thelongest dimension of a pore, e.g., a diameter of a pore having acircular cross section, or the length of the longest cross-sectionalchord that can be constructed across a pore having a non-circularcross-section. In other embodiments, the pore size can refer theshortest dimension of a pore.

Methods for generating porous structures within silk fibroin matrix,e.g., freeze-drying, porogen-leaching method (e.g., salt-leaching), andgas foaming methods, are well known in the art and have been describedin, e.g., U.S. Pat. No. 7,842,780; and US Patent Application Nos: US2010/0279112; and US 2010/0279112, the contents of which areincorporated herein by reference in their entirety.

In some embodiments, the porous silk fibroin matrices are not producedby a porogen-leaching method (e.g., salt-leaching method) as describedin, e.g., U.S. Pat. No. 7,842,780; and US 2010/0279112.

In some embodiments, porous silk fibroin matrices can be produced byfreeze-drying method. See, e.g., U.S. Pat. No. 7,842,780, and US2010/0279112. In such embodiments, the silk fibroin solution placed in anon-stick container can be frozen at sub-zero temperatures, e.g., fromabout −80° C. to about −20° C., for at least about 12 hours, at leastabout 24 hours, or longer, followed by lyophilization. In oneembodiment, the silk fibroin solution can be frozen from one direction.In some embodiments, the silk fibroin solution can contain no salt. Insome embodiments, alcohol such as 15%-25% of methanol or propanol can beadded to the silk fibroin solution.

In certain embodiments, porous silk fibroin matrices can be produced byfreezing the silk fibroin solution at a temperature range between about−1° C. and about −20° C. or between about −5° C. and −10° C., for atleast about 2 days, at least about 3 days or longer, followed bylyophilization for at least about 2 days, at least about 3 days orlonger. See, e.g., U.S. 61/477,486. The freezing temperature and/orduration, and/or lyophilization duration can be adjusted to generate asilk fibroin matrix of different porous structures and/or mechanicalproperties.

In some embodiments, the silk fibroin solution can be exposed to anelectric field, e.g., by applying a voltage to the silk fibroinsolution. The silk fibroin solution that did not change to a gel afterexposure to an electric field can then be placed in a freezer for anextended period of time, e.g., at least about 1 day, at least about 2days, at least about 3 days, at least about 5 days, at least about 6days or longer. The frozen silk fibroin matrix can then be removed fromthe freezer and stored at about room temperature, resulting in a silkfibroin matrix of various porous structures and/or properties.

In some embodiments, silk fibroin matrices described herein can besubjected to a post-treatment that will affect at least one silk fibroinproperty. For example, post-treatment of silk fibroin matrices canaffect silk fibroin properties including β-sheet content, solubility,active agent loading capacity, degradation time, drug permeability orany combinations thereof. Silk post-processing options includecontrolled slow drying (Lu et al., 10 Biomacromolecules 1032 (2009)),water annealing (Jin et al., Water-Stable Silk Films with Reducedβ-Sheet Content, 15 Adv. Funct. Mats. 1241 (2005)), stretching (Demura &Asakura, Immobilization of glucose oxidase with Bombyx mori silk fibroinby only stretching treatment and its application to glucose sensor, 33Biotech & Bioengin. 598 (1989)), compressing, and solvent immersion,including methanol (Hofmann et al., 2006), ethanol (Miyairi et al.,1978), glutaraldehyde (Acharya et al., 2008) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Bayraktar et al., 2005).

In some embodiments, post-treatment of the silk fibroin matrices, e.g.,water-annealing or solvent immersion, can allow controlling the releaseof an active agent from the silk fibroin matrices. In some embodiments,post-treatment of the silk fibroin matrices, e.g., water-annealing orsolvent immersion, can enable modulating the degradation or solubilityproperties of the silk fibroin matrices used in the methods describedherein. In some embodiments, post-treatment of the silk fibroinmatrices, e.g., water-annealing or solvent immersion, can enablemodulating the volume retention properties of the silk fibroin matricesused in the methods described herein.

In some embodiments, the silk fibroin matrices described herein can becoated with at least one layer of a biocompatible and/or biodegradablepolymer described herein, e.g., to modulate the degradation and/orvolume retention properties of the silk fibroin matrices upon injectioninto a tissue to be treated and/or to modulate the rate of active agentsreleased from the silk fibroin matrices. In such embodiments, thebiocompatible and/or biodegradable polymer can comprise at least oneactive agent.

In some embodiments, the silk fibroin matrices described herein can becoated with cell adhesion molecules, e.g., but not limited to,fibronectin, vitronectin, laminin, collagen, any art-recognizedextracellular matrix molecules, and any combinations thereof.

In some embodiments, the silk fibroin matrices described herein can besterilized. Sterilization methods for biomedical devices are well knownin the art, including, but not limited to, gamma or ultravioletradiation, autoclaving (e.g., heat/steam); alcohol sterilization (e.g.,ethanol and methanol); and gas sterilization (e.g., ethylene oxidesterilization).

Further, the silk fibrin matrices described herein can take advantage ofthe many techniques developed to functionalize silk fibroin (e.g.,active agents such as dyes and sensors). See, e.g., U.S. Pat. No.6,287,340, Bioengineered anterior cruciate ligament; WO 2004/000915,Silk Biomaterials & Methods of Use Thereof; WO 2004/001103, SilkBiomaterials & Methods of Use Thereof; WO 2004/062697, Silk FibroinMaterials & Use Thereof; WO 2005/000483, Method for Forming inorganicCoatings; WO 2005/012606, Concentrated Aqueous Silk Fibroin Solution &Use Thereof; WO 2011/005381, Vortex-Induced Silk fibroin Gelation forEncapsulation & Delivery; WO 2005/123114, Silk-Based Drug DeliverySystem; WO 2006/076711, Fibrous Protein Fusions & Uses Thereof in theFormation of Advanced Organic/Inorganic Composite Materials; U.S.Application Pub. No. 2007/0212730, Covalently immobilized proteingradients in three-dimensional porous scaffolds; WO 2006/042287, Methodfor Producing Biomaterial Scaffolds; WO 2007/016524, Method for StepwiseDeposition of Silk Fibroin Coatings; WO 2008/085904, BiodegradableElectronic Devices; WO 2008/118133, Silk Microspheres for Encapsulation& Controlled Release; WO 2008/108838, Microfluidic Devices & Methods forFabricating Same; WO 2008/127404, Nanopatterned Biopolymer Device &Method of Manufacturing Same; WO 2008/118211, Biopolymer PhotonicCrystals & Method of Manufacturing Same; WO 2008/127402, BiopolymerSensor & Method of Manufacturing Same; WO 2008/127403, BiopolymerOptofluidic Device & Method of Manufacturing the Same; WO 2008/127401,Biopolymer Optical Wave Guide & Method of Manufacturing Same; WO2008/140562, Biopolymer Sensor & Method of Manufacturing Same; WO2008/127405, Microfluidic Device with Cylindrical Microchannel & Methodfor Fabricating Same; WO 2008/106485, Tissue-Engineered Silk Organs; WO2008/140562, Electroactive Biopolymer Optical & Electro-Optical Devices& Method of Manufacturing Same; WO 2008/150861, Method for Silk FibroinGelation Using Sonication; WO 2007/103442, Biocompatible Scaffolds &Adipose-Derived Stem Cells; WO 2009/155397, Edible Holographic SilkProducts; WO 2009/100280, 3-Dimensional Silk HydroxyapatiteCompositions; WO 2009/061823, Fabrication of Silk Fibroin PhotonicStructures by Nanocontact Imprinting; WO 2009/126689, System & Methodfor Making Biomaterial Structures.

In an alternative embodiment, the silk fibroin matrices can includeplasmonic nanoparticles to form photothermal elements. This approachtakes advantage of the superior doping characteristics of silk fibroin.Thermal therapy has been shown to aid in the delivery of various agents,see Park et al., Effect of Heat on Skin Permeability, 359 Intl. J.Pharm. 94 (2008). In one embodiment, short bursts of heat on verylimited areas can be used to maximize permeability with minimal harmfuleffects on surrounding tissues. Thus, plasmonic particle-doped silkfibroin matrices can add specificity to thermal therapy by focusinglight to locally generate heat only via the silk fibroin matrices. Insome embodiments, the silk fibroin matrices can include photothermalagents such as gold nanoparticles.

In some embodiments, the silk fibroin matrices used in the methodsdescribed herein can include an amphiphilic peptide. In otherembodiments, the silk fibroin matrices used in the methods describedherein can exclude an amphiphilic peptide. “Amphiphilic peptides”possess both hydrophilic and hydrophobic properties. Amphiphilicmolecules can generally interact with biological membranes by insertionof the hydrophobic part into the lipid membrane, while exposing thehydrophilic part to the aqueous environment. In some embodiment, theamphiphilic peptide can comprise a RGD motif. An example of anamphiphilic peptide is a 23RGD peptide having an amino acid sequence:HOOC-Gly-ArgGly-Asp-Ile-Pro-Ala-Ser-Ser-Lys-Gly-Gly-Gly-Gly-SerArg-Leu-Leu-Leu-Leu-Leu-Leu-Arg-NH2.Other examples of amphiphilic peptides include the ones disclosed in theU.S. Patent App. No.: US 2011/0008406.

Injectable Compositions Comprising a Silk Fibroin Matrix

In another aspect, provided herein is an injectable composition for usein repairing or augmenting a tissue in a subject comprising a compressedsilk fibroin matrix, wherein the compressed silk fibroin matrix expandsupon injection into the tissue, and retains its original expanded volume(e.g., at least about 50% or higher) within the tissue to be repaired oraugmented for a period of time (e.g., at least about 2 weeks, at leastabout 4 weeks, at least about 6 weeks or longer).

As used herein, the term “injectable composition” generally refers to acomposition that can be delivered or administered into a tissue with aminimally invasive procedure. The term “minimally invasive procedure”refers to a procedure that is carried out by entering a subject's bodythrough the skin or through a body cavity or an anatomical opening, butwith the smallest damage possible (e.g., a small incision, injection).In some embodiments, the injectable composition can be administered ordelivered into a tissue by injection. In some embodiments, theinjectable composition can be delivered into a tissue through a smallincision on the skin followed by insertion of a needle, a cannula and/ortubing, e.g., a catheter. Without wishing to be limited, the injectablecomposition can be administered or placed into a tissue by surgery,e.g., implantation.

In some embodiments, the injectable compositions can comprise at leastone active agent described herein.

In some embodiments, the injectable composition can comprise at leastone cell. The term “cells” used herein refers to any cell, prokaryoticor eukaryotic, including plant, yeast, worm, insect and mammalian. Insome embodiments, the cells can be mammalian cells. Mammalian cellsinclude, without limitation; primate, human and a cell from any animalof interest, including without limitation; mouse, rat, hamster, rabbit,dog, cat, domestic animals, such as equine, bovine, murine, ovine,canine, feline, etc. The cells can be a wide variety of tissue typeswithout limitation such as; hematopoietic, neural, mesenchymal,cutaneous, mucosal, stromal, muscle spleen, reticuloendothelial,epithelial, endothelial, hepatic, kidney, gastrointestinal, pulmonary,T-cells etc. Stem cells, embryonic stem (ES) cells, ES-derived cells andstem cell progenitors are also included, including without limitation,hematopoietic, neural, stromal, muscle, cardiovascular, hepatic,pulmonary, and gastrointestinal stem cells and adipose-derived stemcells. In one embodiment, the cells are adipose-derived stem cells. Insome embodiments, the cells can be ex vivo or cultured cells, e.g. invitro. For example, for ex vivo cells, cells can be obtained from asubject, where the subject is healthy and/or affected with a disease.Cells can be obtained, as a non-limiting example, by biopsy or othersurgical means known to those skilled in the art. In some embodiments,adipose cells can be harvested from a subject by conventionalliposuction or aspiration techniques. In such embodiments, the cells canbe derived from a lipoaspirate. In other embodiments, the cells can bederived from a bone-marrow aspirate. Depending on the types of tissuesto be repaired or augmented, cells can be derived from any biologicalfluid or concentrate, e.g., a lipoaspirate or a bone-marrowlipoaspirate. In some embodiments, the injectable composition or thesilk fibroin matrix can be directly delivered with a biological fluid orconcentrate, e.g., a lipoaspirate or a bone-marrow aspirate.

Cells can be obtained from donors (allogenic) or from recipients(autologous). Cells can also be of established cell culture lines, oreven cells that have undergone genetic engineering. Additionally, cellscan be collected from a multitude of hosts including but not limited tohuman autograft tissues, transgenic mammals, or bacterial cultures(possibly for use as a probiotic treatment). In certain embodiments, theinjectable compositions and/or silk fibroin matrices can comprise humanstem cells such as, e.g., mesenchymal stem cells, induced pluripotentstem cells (iPSCs), synovial derived stem cells, embryonic stem cells,adult stem cells, umbilical cord blood cells, umbilical Wharton's jellycells, osteocytes, fibroblasts, neuronal cells, lipocytes, adipocytes,bone marrow cells, assorted immunocytes, precursor cells derived fromadipose tissue, bone marrow derived progenitor cells, peripheral bloodprogenitor cells, stem cells isolated from adult tissue and geneticallytransformed cells or combinations of the above cells; or differentiatedcells such as, e.g., muscle cells, adipose cells.

Stem cells can be obtained with minimally invasive procedures from bonemarrow, adipose tissue, or other sources in the body, are highlyexpandable in culture, and can be readily induced to differentiate intoadipose tissue forming cells after exposure to a well-establishedadipogenic inducing supplement. Cells can be added to the injectablecompositions and/or silk fibroin matrices described herein and culturedin vitro for a period of time prior to administration to a region of thebody, or added to injectable compositions and/or silk fibroin matricesdescribed herein and administered into a region of the body. The cellscan be seeded on the silk fibroin matrices for a short period of time(less than 1 day) just prior to administration, or cultured for a longer(more than 1 day) period to allow for cell proliferation andextracellular matrix synthesis within the seeded matrix prior toadministration.

When utilized as a source of stem cells, adipose tissue can be obtainedby any method known to a person of ordinary skill in the art. Forexample, adipose tissue can be removed from an individual bysuction-assisted lipoplasty, ultrasound-assisted lipoplasty, andexcisional lipectomy. In addition, the procedures can include acombination of such procedures. Suction assisted lipoplasty can bedesirable to remove the adipose tissue from an individual as it providesa minimally invasive method of collecting tissue with minimal potentialfor stem cell damage that can be associated with other techniques, suchas ultrasound assisted lipoplasty. The adipose tissue should becollected in a manner that preserves the viability of the cellularcomponent and that minimizes the likelihood of contamination of thetissue with potentially infectious organisms, such as bacteria and/orviruses.

In some embodiments, preparation of the cell population can requiredepletion of the mature fat-laden adipocyte component of adipose tissue.This is typically achieved by a series of washing and disaggregationsteps in which the tissue is first rinsed to reduce the presence of freelipids (released from ruptured adipocytes) and peripheral blood elements(released from blood vessels severed during tissue harvest), and thendisaggregated to free intact adipocytes and other cell populations fromthe connective tissue matrix. Disaggregation can be achieved using anyconventional techniques or methods, including mechanical force (mincingor shear forces), enzymatic digestion with single or combinatorialproteolytic enzymes, such as collagenase, trypsin, lipase, liberase HIand pepsin, or a combination of mechanical and enzymatic methods. Forexample, the cellular component of the intact tissue fragments can bedisaggregated by methods using collagenase-mediated dissociation ofadipose tissue, similar to the methods for collecting microvascularendothelial cells in adipose tissue, as known to those of skill in theart. Additional methods using collagenase that can be used are alsoknown to those of skill in the art. Furthermore, methods can employ acombination of enzymes, such as a combination of collagenase and trypsinor a combination of an enzyme, such as trypsin, and mechanicaldissociation.

The cell population (processed lipoaspirate) can then be obtained fromthe disaggregated tissue fragments by reducing the presence of matureadipocytes. Separation of the cells can be achieved by buoyant densitysedimentation, centrifugation, elutriation, differential adherence toand elution from solid phase moieties, antibody-mediated selection,differences in electrical charge; immunomagnetic beads, fluorescenceactivated cell sorting (FACS), or other means.

Following disaggregation the active cell population can be washed/rinsedto remove additives and/or by-products of the disaggregation process(e.g., collagenase and newly released free lipid). The active cellpopulation could then be concentrated by centrifugation. In oneembodiment, the cells are concentrated and the collagenase removed bypassing the cell population through a continuous flow spinning membranesystem or the like, such as, for example, the system disclosed in U.S.Pat. Nos. 5,034,135; and 5,234,608, which are incorporated by referenceherein.

In addition to the foregoing, there are many post-wash methods that canbe applied for further purifying the cell population. These include bothpositive selection (selecting the target cells), negative selection(selective removal of unwanted cells), or combinations thereof. Inanother embodiment the cell pellet could be resuspended, layered over(or under) a fluid material formed into a continuous or discontinuousdensity gradient and placed in a centrifuge for separation of cellpopulations on the basis of cell density. In a similar embodiment,continuous flow approaches such as apheresis and elutriation (with orwithout countercurrent) could be used. Adherence to plastic followed bya short period of cell expansion has also been applied in bonemarrow-derived adult stem cell populations. This approach uses cultureconditions to preferentially expand one population while otherpopulations are either maintained (and thereby reduced by dilution withthe growing selected cells) or lost due to absence of required growthconditions. The cells that have been concentrated, cultured and/orexpanded can be incorporated into the silk fibroin matrices and/orinjectable compositions described herein.

In one embodiment, stem cells are harvested, the harvested cells arecontacted with an adipogenic medium for a time sufficient to inducedifferentiation into adipocytes, and the adipocytes are loaded onto abiocompatible matrix which is implanted. In additional embodiments, atleast some of the stem cells can be differentiated into adipocytes sothat a mixture of both cell types is initially present that changes overtime to substantially only adipocytes, with stem cells being present insmall to undetectable quantities. Adipose tissue is fabricated in vivoby the stem cells or prepared ex vivo by the stem cells.

A number of different cell types or combinations thereof can be employedin the injectable compositions, depending upon the types of tissues tobe repaired or augmented. These cell types include, but are not limitedto: smooth muscle cells, skeletal muscle cells, cardiac muscle cells,epithelial cells, endothelial cells, urothelial cells, fibroblasts,myoblasts, chondrocytes, chondroblasts, osteoblasts, osteoclasts,keratinocytes, hepatocytes, bile duct cells, pancreatic islet cells,thyroid, parathyroid, adrenal, hypothalamic, pituitary, ovarian,testicular, salivary gland cells, adipocytes, and precursor cells. Byway of example only, smooth muscle cells and endothelial cells can beemployed when the injectable compositions are used to repair or augmentmuscular and/or vascular tissues, such as vascular, esophageal,intestinal, rectal, or ureteral tissues; chondrocytes can be included ininjectable compositions for cartilaginous tissues; fibroblasts can beincluded in injectable compositions intended to replace and/or enhanceany of the wide variety of tissue types (e.g., skin) that containsextracellular matrix, e.g., collagen; adipocytes can be included ininjectable compositions intended to repair or augment any of the widevariety of adipose tissues. In general, any cells that are found in thenatural tissue can be included in the injectable compositions used forcorresponding tissue. In addition, progenitor cells, such as myoblastsor stem cells, can be included to produce their correspondingdifferentiated cell types.

In some embodiments, the injectable compositions can further comprise apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” refers to apharmaceutically-acceptable material, composition or vehicle foradministration of a silk fibroin matrix, and optionally an active agent.Pharmaceutically acceptable carriers include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents, andisotonic and absorption delaying agents, which are compatible with thesilk fibroin matrices and the activity of the active agent, if any, andare physiologically acceptable to the subject. The pharmaceuticalformulations suitable for injection include sterile aqueous solutions ordispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, cell culture medium, buffers (e.g.,phosphate buffered saline), polyol (for example, glycerol, propyleneglycol, liquid polyethylene glycol, and the like), suitable mixturesthereof. In some embodiments, the pharmaceutical carrier can be abuffered solution (e.g. PBS).

Additionally, various additives which enhance the stability, sterility,and isotonicity of the injectable compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it maybe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. The injectable compositions can also containauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, gelling or viscosity enhancing additives, preservatives, colors,and the like, depending upon the preparation desired. Standard texts,such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation.

Viscosity of the injectable compositions can be maintained at theselected level using a pharmaceutically acceptable thickening agent. Inone embodiment, methylcellulose is used because it is readily andeconomically available and is easy to work with. Other suitablethickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropyl cellulose, carbomer, and the like. Thepreferred concentration of the thickener will depend upon the agentselected, and the desired viscosity for injection. The important pointis to use an amount which will achieve the selected viscosity, e.g.,addition of such thickening agents into some embodiments of theinjectable compositions.

Typically, any additives (in addition to the silk fibroin matricesdescribed herein and/or additional active agents) can be present in anamount of 0.001 to 50 wt % dry weight or in a buffered solution. In someembodiments, the active agent can be present in the order of microgramsto milligrams to grams, such as about 0.0001 to about 5 wt %, about0.0001 to about 1 wt %, about 0.0001 to about 0.05 wt % or about 0.001to about 20 wt %, about 0.01 to about 10 wt %, and about 0.05 to about 5wt %. For any pharmaceutical composition to be administered to a subjectin need thereof, it is preferred to determine toxicity, such as bydetermining the lethal dose (LD) and LD50 in a suitable animal modele.g., rodent such as mouse or rat; and, the dosage of thecomposition(s), concentration of components therein and timing ofadministering the composition(s), which elicit a suitable response. Suchdeterminations do not require undue experimentation from the knowledgeof the skilled artisan.

Active Agents

In some embodiments, the injectable composition and/or the silk fibroinmatrices described herein can further comprise at least one activeagent. The active agent can be mixed, dispersed, or suspended in theinjectable composition, and/or it can be distributed or embedded in thesilk fibroin matrices. In some embodiments, the active agent can bedistributed, embedded or encapsulated in the silk fibroin matrices. Insome embodiments, the active agent can be coated on surfaces of the silkfibroin matrices. In some embodiments, the active agent can be mixedwith the silk fibroin matrices to form an injectable composition. Theterm “active agent” can also encompass combinations or mixtures of twoor more active agents, as described below. Examples of active agentsinclude, but are not limited to, a biologically active agent (e.g., atherapeutic agent), a cosmetically active agent (e.g., an anti-agingagent), a cell attachment agent (e.g., integrin-binding molecules), andany combinations thereof.

The term “biologically active agent” as used herein refers to anymolecule which exerts at least one biological effect in vivo. Forexample, the biologically active agent can be a therapeutic agent totreat or prevent a disease state or condition in a subject. Examples ofbiologically active agents include, without limitation, peptides,peptidomimetics, aptamers, antibodies or a portion thereof,antibody-like molecules, nucleic acids (DNA, RNA, siRNA, shRNA),polysaccharides, enzymes, receptor antagonists or agonists, hormones,growth factors, autogenous bone marrow, antibiotics, antimicrobialagents, small molecules and therapeutic agents. The biologically activeagents can also include, without limitations, anti-inflammatory agents,anesthetics, active agents that stimulate issue formation, and/orhealing and regrowth of natural tissues, and any combinations thereof.

Anti-inflammatory agents can include, but are not limited to, naproxen,sulindac, tolmetin, ketorolac, celecoxib, ibuprofen, diclofenac,acetylsalicylic acid, nabumetone, etodolac, indomethacin, piroxicam,cox-2 inhibitors, ketoprofen, antiplatelet medications, salsalate,valdecoxib, oxaprozin, diflunisal, flurbiprofen, corticosteroids, MMPinhibitors and leukotriene modifiers or combinations thereof.

Agents that increase formation of new tissues and/or stimulates healingor regrowth of native tissue at the site of injection can include, butare not limited to, fibroblast growth factor (FGF), transforming growthfactor-beta (TGF-β, platelet-derived growth factor (PDGF), epidermalgrowth factors (EGFs), connective tissue activated peptides (CTAPs),osteogenic factors including bone morphogenic proteins, heparin,angiotensin II (A-II) and fragments thereof, insulin-like growthfactors, tumor necrosis factors, interleukins, colony stimulatingfactors, erythropoietin, nerve growth factors, interferons, biologicallyactive analogs, fragments, and derivatives of such growth factors, andany combinations thereof.

Anesthetics can include, but are not limited to, those used in caudal,epidural, inhalation, injectable, retrobulbar, and spinal applications,such as bupivacaine, lidocaine, benzocaine, cetacaine, ropivacaine, andtetracaine, or combinations thereof.

In some embodiments, the active agents can be cosmetically activeagents. By the term “cosmetically active agent” is meant a compound thathas a cosmetic or therapeutic effect on the skin, hair, or nails, e.g.,anti-aging agents, anti-free radical agents, lightening agents,whitening agents, depigmenting agents, darkening agents such asself-tanning agents, colorants, anti-acne agents, shine control agents,anti-microbial agents, anti-inflammatory agents, anti-mycotic agents,anti-parasite agents, external analgesics, sun-blocking agents,photoprotectors, antioxidants, keratolytic agents,detergents/surfactants, moisturizers, nutrients, vitamins, energyenhancers, anti-perspiration agents, astringents, deodorants, hairremovers, firming agents, anti-callous agents, muscle relaxants, agentsfor hair, nail, and/or skin conditioning, and any combination thereof.

In one embodiment, the cosmetically active agent can be selected from,but not limited to, the group consisting of hydroxy acids, benzoylperoxide, sulfur resorcinol, ascorbic acid, D-panthenol, hydroquinone,octyl methoxycinnamate, titanium dioxide, octyl salicylate, homosalate,avobenzone, polyphenolics, carotenoids, free radical scavengers,ceramides, polyunsaturated fatty acids, essential fatty acids, enzymes,enzyme inhibitors, minerals, hormones such as estrogens, steroids suchas hydrocortisone, 2-dimethylaminoethanol, copper salts such as copperchloride, coenzyme Q10, lipoic acid, amino acids such a proline andtyrosine, vitamins, lactobionic acid, acetyl-coenzyme A, niacin,riboflavin, thiamin, ribose, electron transporters such as NADH andFADH2, and other botanical extracts such as aloe vera, feverfew, andsoy, and derivatives and mixtures thereof. Examples of vitamins include,but are not limited to, vitamin A, vitamin Bs (such as vitamin B3,vitamin B5, and vitamin B12), vitamin C, vitamin K, and vitamin E, andderivatives thereof.

In one embodiment, the cosmetically active agents can be antioxidants.Examples of antioxidants include, but are not limited to, water-solubleantioxidants such as sulfhydryl compounds and their derivatives (e.g.,sodium metabisulfite and N-acetyl-cysteine), lipoic acid anddihydrolipoic acid, resveratrol, lactoferrin, ascorbic acid, andascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbylpolypeptide). Oil-soluble antioxidants suitable for use in thecompositions described herein can include, but are not limited to,butylated hydroxytoluene, tocopherols (e.g., tocopheryl acetate),tocotrienols, and ubiquinone. Natural extracts containing antioxidantssuitable for use in the injectable compositions described herein caninclude, but not limited to, extracts containing flavonoids andisoflavonoids and their derivatives (e.g., genistein and diadzein), andextracts containing resveratrol. Examples of such natural extractsinclude grape seed, green tea, pine bark, and propolis. Other examplesof antioxidants can be found on pages 1612-13 of the ICI Handbook.

In some embodiments, the active agents can be cell attachment agents.Examples of cell attachment agents include, but are not limited to,hyaluronic acid, collagen, crosslinked hyaluronic acid/collagen, anintegrin-binding molecule, chitosan, elastin, fibronectin, vitronectin,laminin, proteoglycans, any derivatives thereof, any peptide oroligosaccharide variants thereof, and any combinations thereof. As usedherein, the term “oligosaccharide” means a compound comprising at leasttwo or more sugars, selected from the group consisting of glucose,fructose, galactose, xylose and any combinations thereof. In oneembodiment, the oligosaccharide can be selected from the groupconsisting of fructooligosaccharide, galactooligosaccharide,lactosucrose, isomaltulose, glycosyl sucrose, isomaltooligosaccharide,gentioligosaccharide, xylooligosaccharide and any combinations thereof.As used herein, the term “oligosaccharides” includes disaccharides.

In some embodiments, the injectable compositions and/or silk fibroinmatrices can further comprise at least one additional material for softtissue augmentation, e.g., dermal filler materials, including, but notlimited to, poly(methyl methacrylate) microspheres, hydroxylapatite,poly(L-lactic acid), collagen, gelatin, elastin, and glycosaminoglycans,hyaluronic acid, commercial dermal filler products such as BOTOX® (fromAllergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®, RESTYLANE®,JUVEDERM® (from Allergan), SCULPTRA®, PERLANE®, and CAPTIQUE®, and anycombinations thereof.

In some embodiments, the injectable composition and/or silk fibroinmatrices can comprise metallic nanoparticles (e.g., but not limited to,gold nanoparticles), optical molecules (e.g., but not limited to,fluorescent molecules, and/or quantum dots), and any otherart-recognized contrast agent, e.g., for biomedical imaging.

In various embodiments, the injectable compositions can be stored ortransported dried or hydrated.

When the active agents are embedded in the silk fibroin matrices, thebioactivity of the active agents (e.g., at least about 30% of thebioactivity of the active agents) can be stabilized for a period of time(e.g., days, weeks, or months) under specific conditions. Suchconditions can include, but are not limited to, a state-changing cycle(e.g., freeze-thaw cycles), temperatures (e.g., above 0° C.), airpressures, humidity, and light exposure. See U.S. Application Ser. No.61/477,737. Some embodiments of the injectable composition can be storedor transported between about 0° C. and about 60° C., about 10° C. andabout 60° C., or about 15° C. and about 60° C. In these embodiments, theinjectable compositions can be stored or transported at roomtemperatures while the bioactivity of the active agents (e.g., at leastabout 30% of the bioactivity of the active agents) can be stabilized fora period of time, e.g., at least about 3 weeks or longer.

Applications of Injectable Compositions and Silk Fibroin MatricesDescribed Herein

The injectable compositions described herein can be used in a variety ofmedical uses, including, without limitation, fillers for tissue space,templates for tissue reconstruction or regeneration, scaffolds for cellsin tissue engineering applications, or as a vehicle/carrier for drugdelivery. A silk fibroin matrix described herein injected into a tissueto be repaired or augmented can act as a scaffold to mimic theextracellular matrices (ECM) of the body, and/or promote tissueregeneration. The scaffold can serve as both a physical support and/oran adhesive template for cells to proliferate therein. In someembodiments, the silk fibroin matrix can contain no cells. Yet the silkfibroin matrix can be coated with cell attachment agents, e.g.,collagen, and/or chemoattractants, e.g., growth factors, that canattract host cells to the silk fibroin matrix and support the cellproliferation. In some embodiments, the silk fibroin matrix can beseeded with cells prior to administration to a target tissue to berepaired or augmented.

In some embodiments, provided herein are injectable compositions thatcan be used to fill, volumize, and/or regenerate a tissue in needthereof. The injectable compositions can generally be used for tissuefilling or volumizing, soft tissue augmentation, replacement, cosmeticenhancement and/or tissue repair in a subject. Additionally, theinjectable compositions can be used for filling of any tissue void orindentation that are either naturally formed (e.g., aging) or created bysurgical procedure for removal of tissue (e.g., a dermal cyst or a solidtumor), corticosteroid treatment, immunologic reaction resulting inlipoatrophy, tissue damage resulting from impact injuries or therapeutictreatment (e.g., radiotherapy or chemotherapy). The injectablecompositions can also be used to raise scar depressions.

In certain embodiments, the injectable compositions can be used for softtissue augmentation. As used herein, by the term “augmenting” or“augmentation” is meant increasing, filling in, restoring, enhancing orreplacing a tissue. In some embodiments, the tissue can lose itselasticity, firmness, shape and/or volume. In some embodiments, thetissue can be partially or completely lost (e.g., removal of a tissue)or damaged. In those embodiments, the term “augmenting” or“augmentation” can also refer to decreasing, reducing or alleviating atleast one symptom or defect in a tissue (for example, but not limitedto, loss of elasticity, firmness, shape and/or volume in a tissue;presence of a void or an indentation in a tissue; loss of function in atissue) by injecting into the tissue with at least one injectablecomposition described herein. In such embodiments, at least one symptomor defect in a tissue can be decreased, reduced or alleviated by atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80% or higher, as compared to notreatment. In some embodiments, at least one symptom or defect in atissue can be decreased, reduced or alleviated by at least about 90%, atleast about 95%, at least about 97%, or higher, as compared to notreatment. In some embodiments, at least one symptom or defect in atissue can be decreased, reduced or alleviated by 100% (defect-free orthe defect is undetectable by one of skill in the art), as compared tono treatment. In other embodiments, the tissue can be augmented toprevent or delay the onset of defect manifestation in a tissue, e.g.,loss of elasticity, firmness, shape and/or volume in a tissue, or signsof wrinkles. As used herein, the phrase “soft tissue augmentation” isgenerally used in reference to altering a soft tissue structure,including but not limited to, increasing, filling in, restoring,enhancing or replacing a tissue, e.g., to improve the cosmetic oraesthetic appearance of the soft tissue. For example, breastaugmentation (also known as breast enlargement, mammoplasty enlargement,augmentation mammoplasty) alters the size and shape of a woman's breaststo improve the cosmetic or aesthetic appearance of the woman. Examplesof soft tissue augmentation includes, but is not limited to, dermaltissue augmentation; filling of lines, folds, wrinkles, minor facialdepressions, and cleft lips, especially in the face and neck; correctionof minor deformities due to aging or disease, including in the hands andfeet, fingers and toes; augmentation of the vocal cords or glottis torehabilitate speech; dermal filling of sleep lines and expression lines;replacement of dermal and subcutaneous tissue lost due to aging; lipaugmentation; filling of crow's feet and the orbital groove around theeye; breast augmentation; chin augmentation; augmentation of the cheekand/or nose; bulking agent for periurethral support, filling ofindentations in the soft tissue, dermal or subcutaneous, due to, e.g.,overzealous liposuction or other trauma; filling of acne or traumaticscars; filling of nasolabial lines, nasoglabellar lines and intraorallines. In some embodiments, the injectable compositions and/or silkfibroin matrices described herein can be used to treat faciallipodystrophies. In some embodiments, the injectable compositions can beused for breast augmentation and/or reconstruction.

In some embodiments, the injectable compositions can be used for softtissue repair. The term “repair” or “repairing” as used herein, withrespect to a tissue, refers to any correction, reinforcement,reconditioning, remedy, regenerating, filling of a tissue that restoresvolume, shape and/or function of the tissue. In some embodiments“repair” includes full repair and partial repair. For example, thevolume, shape and/or function of a tissue to be repaired can be restoredby at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80% or higher, as comparedto no treatment. In some embodiments, the volume, shape and/or functionof a tissue to be repaired can be restored by at least about 90%, atleast about 95%, at least about 97%, or higher, as compared to notreatment. In some embodiments, the volume, shape and/or function of atissue to be repaired can be restored by 100% (defect-free or the defectis undetectable by one of skill in the art), as compared to notreatment. In various embodiments, the injectable compositions can beused to repair any soft tissues discussed earlier, e.g., breast, skin,and any soft tissues amenable for soft tissue augmentation. In someembodiments, the term “repair” or “repairing” are used hereininterchangeably with the term “regeneration” or “regenerate” when usedin reference to tissue treatment.

In some embodiments, the injectable compositions can be used for softtissue reconstruction. As used herein, the phrase “soft tissuereconstruction” refers to rebuilding a soft tissue structure that wasseverely damaged or lost, e.g., by a dramatic accident or surgicalremoval. For example, breast reconstruction is the rebuilding of abreast, usually in women. Conventional methods of construct anatural-looking breast generally involve using autologous tissue orprosthetic material. In some embodiments, such breast reconstruction caninclude reformation of a natural-looking areola and nipple, wherein suchprocedure can involve the use of implants or relocated flaps of thepatient's own tissue. In some embodiments, administration of injectablecompositions and/or silk fibroin matrices into a soft tissue region tobe reconstructed can maintain the shape and/or size of the reconstructedsoft tissue structure for a period of time, e.g., at least 6 weeks, atleast about 2 months, at least about 3 months or longer.

Without wishing to be bound, some embodiments of the injectablecompositions can be used for hard tissue (e.g., musculoskeletal)augmentation or repair, such as augmentation or repair of bone,cartilage and ligament.

The injectable compositions and silk fibroin matrix described herein canalso be used for filling a tissue located at or near a prostheticimplant, for example, but not limited to, a conventional breast implantor knee replacement implant. In some embodiments, the injectablecompositions and silk fibroin matrices can be used to interface betweena prosthetic implant and a tissue, e.g., to fill a void between theprosthetic implant and the tissue, and/or to prevent the tissue indirect contact with the prosthetic implant. By way of example only,after placing a prosthetic implant (e.g., a breast implant) in asubject, an injectable composition described herein can be introduced ator adjacent to the implant to fill any void between the implant and thetissue (e.g., breast tissue) and/or “sculpt” the tissue for a morenatural look.

In any of the uses described herein, silk fibroin matrix can be combinedwith cells for purposes of a biologically enhanced repair. Cells couldbe collected from a multitude of hosts including but not limited tohuman autograft tissues, or transgenic mammals. More specifically, humancells used can comprise cells selected from stem cells (e.g.,adipocyte-derived stem cells), osteocytes, fibroblasts, lipocytes,assorted immunocytes, cells from lipoaspirate or any combinationsthereof. In some embodiments, the cells can be added after rinsing ofthe silk fibroin matrices themselves. They can be blended into the silkfibroin matrices, carrier solution, or mixture of silk fibroin matricesand carrier solution prior to injection.

In some embodiments, administering the cells (e.g., stem cells orlipoaspirate) with silk fibroin matrices or an injectable compositiondescribed herein can enhance or accelerate host integration and/ortissue formation over time. The cells can be mixed with the silk fibroinmatrix or an injectable composition described herein, or they can beadministered prior to, concurrently with, or after the silk fibroinmatrix or an injectable composition is introduced into a target site.Without wishing to be bound by theory, the cells can secretepro-angiogenic factors and/or growth factors at the target site. As thetissue regenerates or remodels to fill up a void or repair a defect, thesilk fibroin matrix can degrade accordingly. In some embodiments, thesilk fibroin matrix can integrate with the regenerated host tissue.

In addition, active agents such as therapeutic agents, pharmaceuticals,or specific growth factors added to the silk fibroin matrices forpurposes of improved outcome can be introduced at any or a combinationof several points throughout the silk fibroin matrix production process.In some embodiments, these factors can be added to silk fibroin solutionor the accelerant phase prior to drying and solidification, they can besoaked into the silk fibroin matrix during the accelerant rinsingprocess, or they can be coated onto the bulk silk fibroin followingrinsing. In some embodiments, smaller silk fibroin matrices used fortissue repair or augmentation can be cut out from a larger silk fibroinmatrix, before introducing an active agent into the smaller silk fibroinmatrices. For example, an active agent can be soaked into the silkfibroin matrices, coated onto the silk fibroin matrices, or introducedinto a carrier fluid before or after blending with the silk fibroinmatrices.

In some aspects, the injectable composition and silk fibroin matricesdescribed herein can be used as tissue space fillers. In one embodiment,the tissue space filler is a dermal filler. The dermal filler can beused to improve skin appearance or condition, including, but not limitedto, rehydrating the skin, providing increased elasticity to the skin,reducing skin roughness, making the skin tauter, reducing or eliminatingstretch lines or marks, giving the skin better tone, shine, brightness,and/or radiance, reducing or eliminating wrinkles in the skin, providingwrinkle resistance to the skin and replacing loss of soft tissue.

A dermal filler comprising a silk fibroin matrix can be modulated forits hardness and opacity through alteration of silk fibroinconcentration and formulation method. In one embodiment, a dermal fillercan be produced by forming a silk fibroin matrix (or foam), e.g., from asilk fibroin solution of about 1% (w/v) to about 6% (w/v) such that theycan be compressed and injected into a tissue through a needle orcannula. The needle or cannula can have an outer diameter of no largerthan 4 mm, no larger than 3 mm, no larger than 2 mm, no larger than 1mm, no larger than 0.8 mm, no larger than 0.6 mm, no larger than 0.4 mm,no larger than 0.2 mm or no larger than 0.1 mm. In some embodiments, theneedle or cannula gauge can range from 10 to 34, 11 to 34, 12 to 32, or13 to 30. In some embodiments, the size of the needle or cannula can bedetermined to allow for an appropriate extrusion force of at least 40N.Depending on the size of the silk fibroin matrix to be injected, in someembodiments, the size of the needle or cannula can be determined toallow for an appropriate extrusion force of less than 40 N (nominaldeliverable injection force for a human hand).

Accordingly, another aspect provided herein relates to a method ofimproving a condition and/or appearance of skin in a subject in needthereof. Non-limiting examples of a skin condition or and/or appearanceinclude dehydration, lack of skin elasticity, roughness, lack of skintautness, skin stretch line and/or marks, skin paleness, and skinwrinkles. The method comprises injecting an injectable compositiondescribed herein into a dermal region of the subject, wherein theinjection improves the skin condition and/or appearance. For example,improving a skin appearance include, but are not limited to, rehydratingthe skin, providing increased elasticity to the skin, reducing skinroughness, making the skin tauter, reducing or eliminating stretch linesor marks, giving the skin better tone, shine, brightness and/or radianceto reduce paleness, reducing or eliminating wrinkles in the skin, andproviding wrinkle resistance to the skin.

As used herein, the term “dermal region” refers to the region of skincomprising the epidermal-dermal junction and the dermis including thesuperficial dermis (papillary region) and the deep dermis (reticularregion). The skin is composed of three primary layers: the epidermis,which provides waterproofing and serves as a barrier to infection; thedermis, which serves as a location for the appendages of skin; and thehypodermis (subcutaneous adipose layer). The epidermis contains no bloodvessels, and is nourished by diffusion from the dermis. The main type ofcells which make up the epidermis include, but are not limited to,keratinocytes, melanocytes, Langerhans cells and Merkels cells.

The dermis is the layer of skin beneath the epidermis that consists ofconnective tissue and cushions the body from stress and strain. Thedermis is tightly connected to the epidermis by a basement membrane. Italso harbors many mechanoreceptor/nerve endings that provide the senseof touch and heat. It contains the hair follicles, sweat glands,sebaceous glands, apocrine glands, lymphatic vessels and blood vessels.The blood vessels in the dermis provide nourishment and waste removalfrom its own cells as well as from the Stratum basale of the epidermis.The dermis is structurally divided into two areas: a superficial areaadjacent to the epidermis, called the papillary region, and a deepthicker area known as the reticular region.

The papillary region is composed of loose areolar connective tissue. Itis named for its fingerlike projections called papillae that extendtoward the epidermis. The papillae provide the dermis with a “bumpy”surface that interdigitates with the epidermis, strengthening theconnection between the two layers of skin. The reticular region liesdeep in the papillary region and is usually much thicker. It is composedof dense irregular connective tissue, and receives its name from thedense concentration of collagenous, elastic, and reticular fibers thatweave throughout it. These protein fibers give the dermis its propertiesof strength, extensibility, and elasticity. Also located within thereticular region are the roots of the hair, sebaceous glands, sweatglands, receptors, nails, and blood vessels. Stretch marks frompregnancy are also located in the dermis.

The hypodermis is not part of the skin, and lies below the dermis. Itspurpose is to attach the skin to underlying bone and muscle as well assupplying it with blood vessels and nerves. It consists of looseconnective tissue and elastin. The main cell types are fibroblasts,macrophages and adipocytes (the hypodermis contains 50% of body fat).Fat serves as padding and insulation for the body.

In one embodiment, provided herein is a method of treating skindehydration, which comprises injecting to a dermal region suffering fromskin dehydration an injectable composition described herein, e.g.,wherein the composition comprises a silk fibroin matrix, and optionallya carrier and/or an active agent, and wherein the injection of thecomposition rehydrates the skin, thereby treating skin dehydration.

In another embodiment, a method of treating a lack of skin elasticitycomprises injecting to a dermal region suffering from a lack of skinelasticity an injectable composition described herein, e.g., wherein thecomposition comprises a plurality of a silk fibroin matrix, andoptionally a carrier and/or an active agent, and wherein the injectionof the composition increases the elasticity of the skin, therebytreating a lack of skin elasticity.

In yet another embodiment, a method of treating skin roughness comprisesinjecting to a dermal region suffering from skin roughness an injectablecomposition described herein, e.g., wherein the composition comprises asilk fibroin matrix, and optionally a carrier and/or an active agent,and wherein the injection of the composition decreases skin roughness,thereby treating skin roughness.

In still another embodiment, a method of treating a lack of skintautness comprises injecting to a dermal region suffering from a lack ofskin tautness an injectable composition described herein, e.g., whereinthe composition comprises a silk fibroin matrix as described herein, andoptionally a carrier and/or an active agent, and wherein the injectionof the composition makes the skin tauter, thereby treating a lack ofskin tautness.

In a further embodiment, a method of treating a skin stretch line ormark comprises injecting to a dermal region suffering from a skinstretch line or mark an injectable composition described herein, e.g.,wherein the composition comprises a silk fibroin matrix as describedherein, and optionally a carrier and/or an active agent, and wherein theinjection of the composition reduces or eliminates the skin stretch lineor mark, thereby treating a skin stretch line or mark.

In another embodiment, a method of treating skin wrinkles comprisesinjecting to a dermal region suffering from skin wrinkles an injectablecomposition described herein, e.g., wherein the composition comprises asilk fibroin matrix, and optionally a carrier and/or an active agent,and wherein the injection of the composition reduces or eliminates skinwrinkles, thereby treating skin wrinkles.

In yet another embodiment, a method of treating, preventing or delayingthe formation of skin wrinkles comprises injecting to a dermal regionsusceptible to, or showing signs of wrinkles an injectable compositiondescribed herein, e.g., wherein the composition comprises a silk fibroinmatrix, and optionally a carrier and/or an active agent, and wherein theinjection of the composition makes the skin resistant to skin wrinkles,thereby treating, preventing or delaying the formation of skin wrinkles.

The effective amount/size and administration schedule of silk fibroinmatrices injected into a dermal region can be determined by a person ofordinary skill in the art taking into account various factors,including, without limitation, the type of skin condition, the locationof the skin condition, the cause of the skin condition, the severity ofthe skin condition, the degree of relief desired, the duration of reliefdesired, the particular silk fibroin matrix formulation used, the rateof degradation or volume retention of the particular silk fibroin matrixformulation used, the pharmacodynamics of the particular silk fibroinmatrix formulation used, the nature of the other compounds included inthe particular silk fibroin matrix formulation used, the particularcharacteristics, history and risk factors of the individual, such as,e.g., age, weight, general health, and any combinations thereof. In someembodiments, the silk fibroin matrix can be injected into a dermalregion every 3 months, every 6 months, every 9 months, every year, everytwo years or longer.

In another aspect, the injectable compositions can be used as a dermalfiller for dermal bulking to reconstruct or augment a soft tissue bodypart, such as, e.g., a lip, a breast, a breast part such as the nipple,a muscle, or any other soft body part where adipose and/or connectivetissue is used to provide shape, insulation, or other biologicalfunction. In fillers used for these applications, the silk fibroinconcentration and/or the amount of a carrier (e.g., saline) added tosilk fibroin matrix mixture can be adjusted for the relevant constraintsof a given biological environment. For example, silk fibroin matrix forbreast augmentation can be adapted for matrix hardness and volumeretention through alteration of silk fibroin concentration andprocessing method. For example, about 1% (w/v) to about 10% (w/v) silkfibroin concentration, optionally containing an active agent, e.g.,adipose cells such adipose-derived stem cells or cells fromlipoaspirate, can be used to produce the silk fibroin matrix. Carriercontent in the case of saline can be on the order of 0% to 25% (v/v).Other factors such as, e.g., defect type, defect size and needs for aspecific depth of injection of the filler, should be also considered.

Without wishing to be bound, while injection is minimally-invasive,other administration method can be also be used, e.g., implantation,when needed, e.g., to repair or argument a large defect area. Forexample, for dermal injection and lip augmentation, a syringe needlesized 26 g-30 g can be used. In applications involving large quantitiesof filler, e.g., breast reconstruction or augmentation, a larger matrixsize and a larger bore needle or smaller needle gauge such as 23 g-27 gcan be used to administer the filler. In some embodiments, surgery,e.g., implantation, can also be employed to administer large quantitiesof filler and/or to reach a certain depth of tissues.

Accordingly, in a further aspect, provided herein relates to a method ofsoft tissue reconstruction, repair, or augmentation, the methodcomprising administering an injectable composition described herein to asoft tissue region of an individual in need thereof; wherein thecomposition comprises a silk fibroin matrix as described herein, andoptionally an active agent and/or a carrier. Administration methods ofan injectable composition described herein can be determined by anordinary artisan. In some embodiments, the administration method can beinjection. In some embodiments, the administration method can besurgery, e.g., implantation.

While injectable compositions and/or silk fibroin matrices describedherein can be directly applied on a target region (e.g., injection orsurgery), in some embodiments, an injectable composition and/or silkfibroin matrix disclosed herein can also be used to fill an expandableimplantable medical device, such as, e.g., an expandable breast implantshell, which is placed in a defect area. In such embodiments, providedherein is a method of soft tissue reconstruction, repair oraugmentation, the method comprising placing an implantable medicaldevice into a soft tissue region of an individual at the desiredlocation; and expanding the device by filling the device with silkfibroin matrix and/or injectable compositions described herein, whereinexpansion of the medical device by filling it with silk fibroin matrixand/or injectable compositions described herein can reconstruct oraugment the soft tissue.

The silk fibroin matrices or injectable compositions disclosed hereincan be also used in conjunction with interventional radiologyembolization procedures for blocking abnormal blood (artery) vessels(e.g., for the purpose of stopping bleeding) or organs (to stop theextra function e.g. embolization of the spleen for hypersplenism)including uterine artery embolization for percutaneous treatment ofuterine fibroids. Modulation of silk fibroin matrix hardness and volumeretention rate can be done through alteration of silk fibroinconcentration and processing methods as described earlier.

The silk fibroin matrices or injectable compositions disclosed hereincan be used to repair void space in a spine, e.g., created by spine disknucleus removal surgery, to help maintain the normal distance betweenthe adjacent vertebral bodies. In some embodiments, a vertebral discfiller comprising a plurality of silk fibroin matrices can be used torepair void space present in the spine, e.g., between vertebral bodies,and/or in a ruptured spine disk. In such embodiments, a silk fibroinconcentration of about 1% (w/v) to about 10% (w/v) can be used tofabricate the silk fibroin matrix described herein. Accelerant and/oractive agents can also be mixed with silk fibroin matrix and/orinjectable compositions before, during, or after injection into the siteof interest.

The silk fibroin matrix or injectable compositions disclosed herein canbe used to fill up the vitreous cavity to support the eyeball structureand maintain the retina's position. The viscosity of the injectablecomposition described herein can be adjusted for the viscosity ofvitreous fluid in the eye by one of skill in the art.

In some embodiments, the silk fibroin matrix and/or injectablecompositions can be used as a template for tissue reconstruction oraugmentation, e.g., soft tissue reconstruction or augmentation (e.g.,breast augmentation), or even for small bone or cartilage defects suchas fractures. The administration of silk fibroin matrices or injectablecompositions described herein can be used to facilitate cartilage/bonecell ingrowth and proliferation and support collagen matrix depositionthus to improve cartilage/bone repair. In another aspect, prior toadministration, donor cartilage cells can be seeded or mixed with silkfibroin matrices and/or injectable compositions described herein toexpand cell population and thus to promote the development of cartilagetissue. In some embodiments, specific growth factors such as TGF-β orbone morphogenic proteins (BMPs) which support cartilage or bone tissueformation, respectively, can be added into silk fibroin matrices.

In another embodiment, the silk fibroin matrices and/or injectablecompositions described herein can be used for facial plastic surgery,such as, e.g., nose reconstruction. The reconstruction strategydiscussed above for repairing a cartilage/bone defect can also beapplicable for facial plastic surgery.

In some embodiments, the silk fibroin matrices and/or injectablecompositions described herein can be used as scaffolds to support cellgrowth for tissue engineering. For example, the silk fibroin matricesand/or injectable compositions described herein can be administered intoan incision or wound site to promote wound healing or wound disclosure.The methods generally comprise administering an injectable compositionor silk fibroin matrices described herein, at the wound or incision siteand allowing the wound or incision to heal while the silk fibroin matrixis eroded or absorbed in the body and is replaced with the individual'sown viable tissue. The methods can further comprise seeding the silkfibroin matrices or mixing the injectable composition with viablecellular material, either from the individual or from a donor, prior toor during administration.

In another aspect, the injectable composition comprising a silk fibroinmatrix can be used, directly or indirectly, in methods of repairing,augmenting, or reconstructing a tissue in a subject, e.g., augmenting orreconstructing the breast of a human being. In some embodiments, theinjectable compositions or a silk fibroin matrix can be directly placedinto a tissue (e.g., a breast tissue) to be repaired or augmented, e.g.,by injection. The injectable compositions or a silk fibroin matrix canbe injected into a tissue (e.g., a breast tissue) every 6 months, everyyear, every 2 years, every 3 years, or longer. In other embodiments, theinjectable compositions or a silk fibroin matrix can be used to enhancesupport of a conventional tissue implant, e.g., by enhancing support ofthe lower pole position of a breast implant. In alternative embodiments,the method can generally comprise administering an injectablecomposition and/or a silk fibroin matrix near or in proximity to atissue implant, for example, a conventional breast implant, and seedingthe injectable composition and/or silk fibroin matrix with viablecellular material prior to or during administration. In yet anotherembodiment, an injectable composition and/or a silk fibroin matrix canbe used to partially or completely cover a tissue implant (e.g., abreast implant) to provide a beneficial interface with host tissue andto reduce the potential for malpositioning or capsular contracture.

In some embodiments, the silk fibroin matrix and/or injectablecompositions described herein can be used as fillers to promote orsupport adipogenesis, e.g., to treat facial lipodystrophies. In suchembodiments, the injectable compositions and/or silk fibroin matricescan be seeded or mixed with adipose-associated cells, suchadipose-derived stem cells or lipoaspirate, prior to or concurrentlywith the injection to a target area suffering from faciallipodystrophies in a subject. In some embodiments, the silk fibroinmatrix can be injected every 3 months, every 6 months, every 9 months,every year, or every two years or longer, to maintain the treatment.

In still another embodiment, the silk fibroin matrices and/or injectablecompositions described herein can be used as the scaffold for cellsuseful for peripheral nerve repair. Silk fibroin matrices can bedelivered (e.g., via injection) to the location of the nerve defect withor without additional device to aid the connection to the nerve ends.For such purpose, specific growth factors such as nerve growth factor(NGF), which supports nerve regeneration can be added into injectablecompositions and/or mixed with silk fibroin matrices prior to or duringadministration. In such embodiments, softer silk fibroin matrices, e.g.using a silk fibroin concentration of about 0.5 (w/v) to about 3% (w/v),can be used. Depending on the brain microenvironment, harder silkfibroin matrices can also be used. The silk fibroin matrices and/orinjectable compositions can be infused with or added with appropriatetherapeutic factors according to the methods described above.

Any cells described herein can be seeded upon a surface of silk fibroinmatrices described herein. For example, silk fibroin matrices can besubmersed in an appropriate growth medium for the cells of interest, andthen directly exposed to the cells. The cells are allowed to proliferateon the surface and interstices of the silk fibroin matrices. The silkfibroin matrices are then removed from the growth medium, washed ifnecessary, and administered. Alternatively, the cells can be placed in asuitable buffer or liquid growth medium and drawn through silk fibroinmatrices by using vacuum filtration. Cells can also be admixed with silkfibroin solution prior to forming silk fibroin matrices, capturing atleast some of the cells within the silk fibroin matrices. In anotherembodiment, the cells of interest can be dispersed into an appropriatesolution (e.g., a growth medium or buffer) and then sprayed onto silkfibroin matrices. For example, electro-spraying involves subjecting acell-containing solution with an appropriate viscosity and concentrationto an electric field sufficient to produce a spray of small chargeddroplets of solution that contain cells.

In some embodiments, the silk fibroin matrices or injectablecompositions comprising at least one active agent can be used as aplatform for drug delivery. For example, the silk fibroin matrices canbe formed with a pharmaceutical agent either entrained in or bound tothe matrices and then administered into the body (e.g., injection,implantation or even oral administration). In some embodiments, anactive agent can be mixed with silk fibroin matrices and/or injectablecompositions and then administered into the body (e.g., injection,implantation or even oral administration). For extended or sustainedrelease, silk fibroin matrices can manipulated, e.g., to modulate itsbeta-sheet content, for its volume retention and/or degradation rate. Tofurther control the drug release profile, the pharmaceutically-activedrug-containing silk fibroin matrices can be mixed with an additionalsilk fibroin gel phase acting as a carrier either with or without aviscosity inducing component, a surfactant, and/or an included lubricantfluid like saline. The therapeutic-bound silk fibroin matrices can alsobe further crosslinked to enhance the stability to extend the releaseperiod. In an alternative approach, silk fibroin matrices can be mixedwith other polymers, for examples, hyaluronic acid, to prolong therelease of certain growth factors or cytokines and to stabilize thefunctionality. Furthermore, the silk fibroin matrices and/or injectablecompositions can also be used for coating coaxial drug delivery systems,e.g., by spraying.

As used herein, the term “sustained release” refers to the release of apharmaceutically-active drug over a period of about seven days or more.In aspects of this embodiment, a drug delivery platform comprising thesilk fibroin matrices and/or injectable compositions releases apharmaceutically-active drug over a period of, e.g., at least about 7days after administration, at least about 15 days after administration,at least about 30 days after administration, at least about 45 daysafter administration, at least about 60 days after administration, atleast about 75 days after administration, or at least about 90 daysafter administration.

As used herein, the term “extended release” refers to the release of apharmaceutically-active drug over a period of time of less than aboutseven days. In such embodiments, a drug delivery platform comprising thesilk fibroin matrix and/or injectable compositions described herein canrelease a pharmaceutically-active drug over a period of, e.g., about 1day after administration, about 2 days after administration, about 3days after administration, about 4 days after administration, about 5days after administration, or about 6 days after administration.

Depending on the formulation and processing methods of the silk fibroinmatrices and the associated applications, the injectable compositions orsilk fibroin matrices can be administered (e.g., by injection)periodically, for example, every 3 months, every 4 months, every 5months, every 6 months, every 7 months, every 8 months, every 9 months,every 10 months, every 11 months, every year, every 2 years or longer.In some embodiments, the injectable compositions or silk fibroinmatrices can be administered once to a tissue to be repaired oraugmented, and the tissue can regenerate over time to replace the silkfibroin matrices.

In some embodiments of any of the applications described herein, theinjectable compositions or silk fibroin matrices can be at leastpartially dry when administered in a tissue to be repaired or augmented.In some embodiments, the injectable compositions or silk fibroinmatrices can be dried (e.g., in the absence of a carrier) whenadministered in a tissue to be repaired or augmented.

In some embodiments of any of the applications described herein, theinjectable compositions or silk fibroin matrices can be at leastpartially hydrated when administered in a tissue to be repaired oraugmented. In some embodiments, the injectable compositions or silkfibroin matrices can be hydrated (e.g., in the presence of a carrier,e.g., a buffered solution and/or lipoaspirate) when administered in atissue to be repaired or augmented.

In some embodiments of any of the applications described herein, theinjectable compositions or silk fibroin matrices can be injectedsubcutaneously, submuscularly, or intramuscularly.

In some embodiments, the methods and/or compositions described hereincan be used in the dermal region. In some embodiments, the methodsand/or compositions described herein can be used in the epidermal layer,dermal layer, hypodermis layer, or any combinations thereof.

Delivery Devices and Kits Comprising Silk Fibroin Matrices

Delivery devices comprising an injectable composition or silk fibroinmatrices described herein are also provided herein. Delivery devices canbe any conventional delivery device used for injection purposes, e.g., asyringe, or a custom-made delivery device, such as an injection gun.Accordingly, a further aspect provided herein is an injection devicecomprising an injectable composition or a silk fibroin matrix.

In some embodiments, the delivery device can further comprise a tubularstructure for introducing the silk fibroin matrix into a tissue to berepaired or augmented. In some embodiments, the tubular structure can betapered. For example, the tapered tubular structure can comprise aconical interior space. Examples of the tubular structures can include,without limitations, a needle, a cannula, a catheter, any art-recognizedinjection applicator, and any combinations thereof.

In some embodiments, the delivery device can further comprise amechanical element (e.g., an elongated structure such as a ramrod) tofacilitate the exit of the compressed silk fibroin matrix through thetubular structure.

In various embodiments, the delivery device can include an injectioncarrier, e.g., a buffered solution.

In various embodiments, the delivery device (e.g., a syringe) caninclude an anesthetic.

Further provided herein is a kit comprising one embodiment of aninjectable composition or silk fibroin matrix packaged in a deliveryapplicator, such as a catheter, a needle or a cannula. In someembodiments, a local anesthetic can be blended with the injectablecomposition or silk fibroin matrix. In alternative embodiments, a localanesthetic can be packaged in a separate container. For example, it isdesirable to apply a local anesthetic to a target tissue to be treatedprior to further treatment. An exemplary anesthetic includes, but is notlimited to, lidocane. Dependent upon application, the kit can includesyringes sizes from 0.5 mL to 60 mL, where applications requiring largervolumes (e.g., bone fillers, disc fillers) are supplied in a larger sizesyringe. Additionally, needle gauge can adjusted according to injectionsite with an acceptable range of 10 g to 30 g needles. For example, 10 gto 20 g needles can be used for intradermal injections.

In some embodiments, the kit can further comprise a plurality ofdelivery devices preloaded with an injectable composition or silkfibroin matrices described herein. Each delivery device can beindividually packaged.

In some embodiments, the kit can further comprise a container containinga buffered solution or an injection carrier.

In some embodiments, the kit can further comprise at least oneadditional empty syringe. In some embodiments, the kit can furthercomprise at least one additional needle. In some embodiments, the kitcan further comprise at least one catheter or cannula.

Embodiments of the Various Aspects Described Herein can be Illustratedby the Following Numbered Paragraphs.

-   -   1. A method for repairing or augmenting a tissue in a subject        comprising: placing into the tissue to be repaired or augmented        a composition comprising a compressed silk fibroin matrix,        wherein the compressed silk fibroin matrix expands upon        placement into the tissue, and retains at least about 1% of its        original expanded volume within the tissue for at least about 2        weeks.    -   2. The method of paragraph 1, wherein the compressed silk        fibroin matrix expands in volume by at least about 2-fold,        relative to the volume of the compressed silk fibroin matrix.    -   3. The method of paragraph 1 or 2, wherein the silk fibroin        matrix retains at least about 50% of its original expanded        volume within the tissue for at least about 2 weeks, at least        about 6 weeks, or at least about 3 months.    -   4. The method of any of paragraphs 1-3, wherein the silk fibroin        matrix retains at least about 50% of its original expanded        volume within the tissue for at least about 6 months.    -   5. The method of any of paragraphs 1-4, wherein the silk fibroin        matrix retains at least about 60% of its original expanded        volume within the tissue for at least about 6 weeks.    -   6. The method of paragraph 5, wherein the silk fibroin matrix        retains at least about 70% of its original expanded volume        within the tissue for at least about 6 weeks.    -   7. The method of paragraph 6, wherein the silk fibroin matrix        retains at least about 80% of its original expanded volume        within the tissue for at least about 6 weeks.    -   8. The method of any of paragraphs 1-7, wherein the silk fibroin        matrix retains at least about 70% of its original expanded        volume within the tissue for at least 3 months.    -   9. The method of any of paragraphs 1-8, wherein the silk fibroin        matrix is adapted to degrade no more than 50% of its original        expanded volume in at least about 6 weeks.    -   10. The method of paragraph 9, wherein the silk fibroin matrix        is adapted to degrade no more than 50% of its original expanded        volume in at least about 3 months.    -   11. The method of any of paragraphs 1-10, wherein the silk        fibroin matrix is adapted to degrade no more than 30% of its        original expanded volume in at least about 6 weeks.    -   12. The method of paragraph 11, wherein the silk fibroin matrix        is adapted to degrade no more than 10% of its original expanded        volume in at least about 6 weeks.    -   13. The method of any of paragraphs 1-12, wherein the silk        fibroin matrix is adapted to degrade no more than 30% of its        original expanded volume in at least about 3 months.    -   14. The method of any of paragraphs 1-13, wherein the silk        fibroin matrix is porous.    -   15. The method of paragraph 14, wherein the porous silk fibroin        matrix has a porosity of at least about 1%, at least about 5%,        at least about 10%, at least about 15%, or at least about 30%.    -   16. The method of paragraph 15, wherein the porous silk fibroin        matrix has a porosity of at least about 50%.    -   17. The method of paragraph 16, wherein the porous silk fibroin        matrix has a porosity of at least about 70%.    -   18. The method of any of paragraphs 14-17, wherein the pores        have a size of about 1 μm to about 1500 μm.    -   19. The method of paragraph 18, wherein the pores have a size of        about 50 μm to about 650 μm.    -   20. The method of any of paragraphs 1-19, wherein the silk        fibroin matrix is formed from a silk fibroin solution of about        0.1% w/v to about 30% w/v.    -   21. The method of paragraph 20, wherein the silk fibroin        solution of about 0.5% w/v to about 10% w/v.    -   22. The method of paragraph 21, wherein the silk fibroin        solution is about 1% w/v to about 6% w/v.    -   23. The method of any of paragraphs 1-22, wherein the silk        fibroin matrix is freezer-processed.    -   24. The method of any of paragraphs 1-23, wherein the silk        fibroin matrix is a silk fibroin foam.    -   25. The method of any of paragraphs 1-24, wherein the        composition or the silk fibroin matrix further comprises at        least one active agent.    -   26. The method of paragraph 25, wherein the at least one active        agent is a biologically active agent, a cosmetically active        agent, a cell attachment agent, or any combinations thereof.    -   27. The method of paragraph 26, wherein the biologically active        agent is selected from the group consisting of a therapeutic        agent, an anesthetic, a cell growth factor, a peptide, a        peptidomimetic, an antibody or a portion thereof, an        antibody-like molecule, nucleic acid, a polysaccharide, and any        combinations thereof.    -   28. The method of paragraph 26, wherein the cell attachment        agent is selected from the group consisting of hyaluronic acid,        collagen, crosslinked hyaluronic acid/collagen, an        integrin-binding molecule, chitosan, elastin, fibronectin,        vitronectin, laminin, proteoglycans, any derivatives thereof,        any peptide or oligosaccharide variants thereof, and any        combinations thereof.    -   29. The method of paragraph 26, wherein the cosmetically active        agent is selected from the group consisting of an anti-aging        agent, an anti-free radical agent, an anti-oxidant, a hydrating        agent, a whitening agent, a colorant, a depigmenting agent, a        sun-blocking agent, a muscle relaxant, and any combinations        thereof.    -   30. The method of any of paragraphs 1-29, wherein the        composition further comprises a cell.    -   31. The method of paragraph 30, wherein the cell is a stem cell.    -   32. The method of any of paragraphs 1-31, wherein the        composition further comprises a biological fluid or concentrate.    -   33. The method of paragraph 32, wherein the biological fluid or        concentrate is lipoaspirate, bone marrow aspirate, or any        combinations thereof.    -   34. The method of any of paragraphs 1-33, wherein the        composition or the silk fibroin matrix further comprises a        hydrogel.    -   35. The method of any of paragraphs 1-34, wherein the        composition or the silk fibroin matrix further comprises a        dermal filler material.    -   36. The method of paragraph 35, wherein the dermal filler        material is selected from the group consisting of poly(methyl        methacrylate) microspheres, hydroxylapatite, poly(L-lactic        acid), hyaluronic acid, collagen, gelatin, and any combinations        thereof.    -   37. The method of any of paragraphs 1-36, wherein the        composition further comprises a carrier.    -   38. The method of any of paragraphs 1-37, wherein the silk        fibroin matrix excludes an amphiphilic peptide.    -   39. The method of paragraph 38, wherein the amphiphilic peptide        comprises a RGD motif.    -   40. The method of any of paragraphs 1-39, wherein the placement        of the compressed silk fibroin matrix is performed by injection.    -   41. The method of paragraph 40, wherein the injection is        performed subcutaneously, submuscularly, or intramuscularly.    -   42. The method of any of paragraphs 1-41, wherein the tissue is        a soft tissue.    -   43. The method of paragraph 42, wherein the soft tissue is        selected from the group consisting of a tendon, a ligament,        skin, a breast tissue, a fibrous tissue, a connective tissue, a        muscle, and any combinations thereof.    -   44. The method of paragraph 43, wherein the soft tissue is skin.    -   45. The method of paragraph 44, wherein the soft tissue is a        breast tissue.    -   46. The method of any of paragraphs 1-45, wherein the subject is        a mammalian subject.    -   47. The method of paragraph 46, wherein the mammalian subject is        a human.    -   48. The method of any of paragraphs 1-47, wherein the silk        fibroin matrix is compressed by loading the silk fibroin matrix        into an interior space of a delivery applicator, wherein the        interior space has a volume smaller than the volume of the silk        fibroin matrix in an uncompressed state.    -   49. The method of paragraph 48, wherein the delivery applicator        comprises a needle, a cannula, a catheter, or any combinations        thereof.    -   50. An injectable composition for use in repairing or augmenting        a tissue in a subject, comprising a compressed silk fibroin        matrix, wherein the compressed silk fibroin matrix expands upon        injection into the tissue, and retains at least about 1% of its        original expanded volume within the tissue for at least about 2        weeks.    -   51. The composition of paragraph 50, wherein the compressed silk        fibroin matrix expands in volume by at least about 2-fold,        relative to the volume of the compressed silk fibroin matrix.    -   52. The composition of paragraph 50 or 51, wherein the        compressed silk fibroin matrix excludes an amphiphilic peptide.    -   53. The composition of paragraph 52, wherein the amphiphilic        peptide comprises a RGD motif.    -   54. The composition of any of paragraphs 50-53, wherein the silk        fibroin matrix retains at least about 50% of its original        expanded volume within the tissue for at least about 2 weeks, at        least about 6 weeks, or at least about 3 months.    -   55. The composition of any of paragraphs 50-54, wherein the silk        fibroin matrix retains at least about 50% of its original        expanded volume within the tissue for at least about 6 months.    -   56. The composition of any of paragraphs 50-55, wherein the silk        fibroin matrix retains at least about 60% of its original        expanded volume within the tissue for at least about 6 weeks.    -   57. The composition of paragraph 56, wherein the silk fibroin        matrix retains at least about 70% of its original expanded        volume within the tissue for at least about 6 weeks.    -   58. The composition of paragraph 57, wherein the silk fibroin        matrix retains at least about 80% of its original expanded        volume within the tissue for at least about 6 weeks.    -   59. The composition of any of paragraphs 50-58, wherein the silk        fibroin matrix retains at least about 70% of its original        expanded volume within the tissue for at least 3 months.    -   60. The composition of any of paragraphs 50-59, wherein the silk        fibroin matrix is adapted to degrade no more than 50% of its        original expanded volume in at least about 6 weeks.    -   61. The composition of paragraph 60, wherein the silk fibroin        matrix is adapted to degrade no more than 50% of its original        expanded volume in at least about 3 months.    -   62. The composition of any of paragraphs 50-62, wherein the silk        fibroin matrix is adapted to degrade no more than 30% of its        original expanded volume in at least about 6 weeks.    -   63. The composition of paragraph 62, wherein the silk fibroin        matrix is adapted to degrade no more than 10% of its original        expanded volume in at least about 6 weeks.    -   64. The composition of any of paragraphs 50-63, wherein the silk        fibroin matrix is adapted to degrade no more than 30% of its        original expanded volume in at least about 3 months.    -   65. The composition of any of paragraphs 50-64, wherein the silk        fibroin matrix is porous.    -   66. The composition of paragraph 65, wherein the porous silk        fibroin matrix has a porosity of at least about 1%, at least        about 5%, at least about 10%, at least about 15%, or at least        about 30%.    -   67. The composition of paragraph 66, wherein the porous silk        fibroin matrix has a porosity of at least about 50%.    -   68. The composition of paragraph 67, wherein the porous silk        fibroin matrix has a porosity of at least about 70%.    -   69. The composition of any of paragraphs 65-68, wherein the        pores have a size of about 1 μm to about 1500 μm.    -   70. The composition of paragraph 69, wherein the pores have a        size of about 50 μm to about 650 μm.    -   71. The composition of any of paragraphs 50-70, wherein the silk        fibroin matrix is formed from a silk fibroin solution of about        0.1% w/v to about 30% w/v.    -   72. The composition of paragraph 71, wherein the silk fibroin        solution of about 0.5% w/v to about 10% w/v.    -   73. The composition of paragraph 72, wherein the silk fibroin        solution is about 1% w/v to about 6% w/v.    -   74. The composition of any of paragraphs 50-73, wherein the silk        fibroin matrix is freezer-processed.    -   75. The composition of any of paragraphs 50-74, wherein the silk        fibroin matrix is a silk fibroin foam.    -   76. The composition of any of paragraphs 50-75, wherein the        injectable composition or the silk fibroin matrix further        comprises at least one active agent.    -   77. The composition of paragraph 76, wherein the at least one        active agent is a biologically active agent, a cosmetically        active agent, a cell attachment agent, or any combinations        thereof.    -   78. The composition of paragraph 77, wherein the biologically        active agent is selected from the group consisting of a        therapeutic agent, an anesthetic, a cell growth factor, a        peptide, a peptidomimetic, an antibody or a portion thereof, an        antibody-like molecule, nucleic acid, a polysaccharide, and any        combinations thereof.    -   79. The composition of paragraph 77, wherein the cell attachment        agent is selected from the group consisting of hyaluronic acid,        collagen, crosslinked hyaluronic acid/collagen, an        integrin-binding molecule, chitosan, elastin, fibronectin,        vitronectin, laminin, proteoglycans, any derivatives thereof,        any peptide or oligosaccharide variants thereof, and any        combinations thereof.    -   80. The composition of paragraph 77, wherein the cosmetically        active agent is selected from the group consisting of an        anti-aging agent, an anti-free radical agent, an anti-oxidant, a        hydrating agent, a whitening agent, a colorant, a depigmenting        agent, a sun-blocking agent, a muscle relaxant, and any        combinations thereof.    -   81. The composition of any of paragraphs 50-80, further        comprising a cell.    -   82. The composition of paragraph 81, wherein the cell is a stem        cell.    -   83. The composition of any of paragraphs 50-82, further        comprising a biological fluid or concentrate.    -   84. The composition of paragraph 83, wherein the biological        fluid or concentrate is lipoaspirate, bone marrow aspirate, or        any combinations thereof.    -   85. The composition of any of paragraphs 50-84, wherein the        injectable composition or the silk fibroin matrix further        comprises a hydrogel.    -   86. The composition of any of paragraphs 50-85, wherein the        injectable composition or the silk fibroin matrix further        comprises a dermal filler material.    -   87. The composition of paragraph 86, wherein the dermal filler        material is selected from the group consisting of poly(methyl        methacrylate) microspheres, hydroxylapatite, poly(L-lactic        acid), hyaluronic acid, collagen, gelatin and any combinations        thereof.    -   88. The composition of any of paragraphs 50-87, wherein the        injectable composition further comprises a carrier.    -   89. The composition of any of paragraphs 50-88, wherein the        compressed silk fibroin matrix has a volume of about 10% to        about 90% of its original volume before compression.    -   90. The composition of any of paragraphs 50-88, wherein the        compressed silk fibroin matrix has a volume of no more than 70%        of its original volume before compression.    -   91. A delivery device comprising an injectable composition of        any of paragraphs 50-90.    -   92. The delivery device of paragraph 91, further comprising a        tubular structure for introducing the injectable composition        into a tissue to be repaired or augmented.    -   93. The delivery device of paragraph 92, wherein the tubular        structure is tapered.    -   94. The delivery device of paragraph 93, wherein the tapered        tubular structure comprises a conical interior space.    -   95. The delivery device of any of paragraphs 91-94, wherein the        tubular structure is a needle, a cannula, a catheter, or any        combinations thereof.    -   96. The delivery device of any of paragraphs 91-95, further        comprising a mechanical element to facilitate the exit of the        compressed silk fibroin matrix through the tubular structure.    -   97. The delivery device of any of paragraphs 91-96, further        comprising an injection carrier.

SOME SELECTED DEFINITIONS OF TERMS

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Incertain embodiments of the aspects described herein, the subject is amammal, e.g., a primate, e.g., a human. A subject can be male or female.Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of tissuerepair, regeneration and/or reconstruction. In addition, the methods andcompositions described herein can be used to treat domesticated animalsand/or pets.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below or above a reference level. The term refers to statisticalevidence that there is a difference. It is defined as the probability ofmaking a decision to reject the null hypothesis when the null hypothesisis actually true. The decision is often made using the p-value.

As used herein, the terms “proteins” and “peptides” are usedinterchangeably herein to designate a series of amino acid residuesconnected to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and “peptide”,which are used interchangeably herein, refer to a polymer of proteinamino acids, including modified amino acids (e.g., phosphorylated,glycated, etc.) and amino acid analogs, regardless of its size orfunction. Although “protein” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Theterm “peptide” as used herein refers to peptides, polypeptides, proteinsand fragments of proteins, unless otherwise noted. The terms “protein”and “peptide” are used interchangeably herein when referring to a geneproduct and fragments thereof. Thus, exemplary peptides or proteinsinclude gene products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants,fragments, and analogs of the foregoing.

The term “nucleic acids” used herein refers to polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA), polymers thereof in either single- or double-stranded form.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides, which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer, et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka, et al., J. Biol. Chem. 260:2605-2608(1985), and Rossolini, et al., Mol. Cell. Probes 8:91-98 (1994)). Theterm “nucleic acid” should also be understood to include, asequivalents, derivatives, variants and analogs of either RNA or DNA madefrom nucleotide analogs, and, single (sense or antisense) anddouble-stranded polynucleotides.

The term “short interfering RNA” (siRNA), also referred to herein as“small interfering RNA” is defined as an agent which functions toinhibit expression of a target gene, e.g., by RNAi. An siRNA can bechemically synthesized, it can be produced by in vitro transcription, orit can be produced within a host cell. siRNA molecules can also begenerated by cleavage of double stranded RNA, where one strand isidentical to the message to be inactivated. The term “siRNA” refers tosmall inhibitory RNA duplexes that induce the RNA interference (RNAi)pathway. These molecules can vary in length (generally 18-30 base pairs)and contain varying degrees of complementarity to their target mRNA inthe antisense strand. Some, but not all, siRNA have unpaired overhangingbases on the 5′ or 3′ end of the sense 60 strand and/or the antisensestrand. The term “siRNA” includes duplexes of two separate strands, aswell as single strands that can form hairpin structures comprising aduplex region.

The term “shRNA” as used herein refers to short hairpin RNA whichfunctions as RNAi and/or siRNA species but differs in that shRNA speciesare double stranded hairpin-like structure for increased stability. Theterm “RNAi” as used herein refers to interfering RNA, or RNAinterference molecules are nucleic acid molecules or analogues thereoffor example RNA-based molecules that inhibit gene expression. RNAirefers to a means of selective post-transcriptional gene silencing. RNAican result in the destruction of specific mRNA, or prevents theprocessing or translation of RNA, such as mRNA.

The term “enzymes” as used here refers to a protein molecule thatcatalyzes chemical reactions of other substances without it beingdestroyed or substantially altered upon completion of the reactions. Theterm can include naturally occurring enzymes and bioengineered enzymesor mixtures thereof. Examples of enzyme families include kinases,dehydrogenases, oxidoreductases, GTPases, carboxyl transferases, acyltransferases, decarboxylases, transaminases, racemases, methyltransferases, formyl transferases, and α-ketodecarboxylases.

As used herein, the term “aptamers” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence capable of specifically recognizing a selectednon-oligonucleotide molecule or group of molecules. In some embodiments,the aptamer recognizes the non-oligonucleotide molecule or group ofmolecules by a mechanism other than Watson-Crick base pairing or triplexformation. Aptamers can include, without limitation, defined sequencesegments and sequences comprising nucleotides, ribonucleotides,deoxyribonucleotides, nucleotide analogs, modified nucleotides andnucleotides comprising backbone modifications, branchpoints andnonnucleotide residues, groups or bridges. Methods for selectingaptamers for binding to a molecule are widely known in the art andeasily accessible to one of ordinary skill in the art.

As used herein, the term “antibody” or “antibodies” refers to an intactimmunoglobulin or to a monoclonal or polyclonal antigen-binding fragmentwith the Fc (crystallizable fragment) region or FcRn binding fragment ofthe Fc region. The term “antibodies” also includes “antibody-likemolecules”, such as fragments of the antibodies, e.g., antigen-bindingfragments. Antigen-binding fragments can be produced by recombinant DNAtechniques or by enzymatic or chemical cleavage of intact antibodies.“Antigen-binding fragments” include, inter alia, Fab, Fab′, F(ab′)2, Fv,dAb, and complementarity determining region (CDR) fragments,single-chain antibodies (scFv), single domain antibodies, chimericantibodies, diabodies, and polypeptides that contain at least a portionof an immunoglobulin that is sufficient to confer specific antigenbinding to the polypeptide. Linear antibodies are also included for thepurposes described herein. The terms Fab, Fc, pFc′, F(ab′) 2 and Fv areemployed with standard immunological meanings (Klein, Immunology (JohnWiley, New York, N.Y., 1982); Clark, W. R. (1986) The ExperimentalFoundations of Modern Immunology (Wiley & Sons, Inc., New York); andRoitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell ScientificPublications, Oxford)). Antibodies or antigen-binding fragments specificfor various antigens are available commercially from vendors such as R&DSystems, BD Biosciences, e-Biosciences and Miltenyi, or can be raisedagainst these cell-surface markers by methods known to those skilled inthe art.

As used herein, the term “Complementarity Determining Regions” (CDRs;i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e. about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e. about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop.

The expression “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

The expression “single-chain Fv” or “scFv” antibody fragments, as usedherein, is intended to mean antibody fragments that comprise the VH andVL domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. (Plückthun, ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994)).

The term “diabodies,” as used herein, refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy-chainvariable domain (VH) Connected to a light-chain variable domain (VL) inthe same polypeptide chain (VH-VL). By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites. (EP 404,097; WO 93/11161; Hollinger etah, Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993)).

As used herein, the term “small molecules” refers to natural orsynthetic molecules including, but not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, aptamers, nucleotides, nucleotide analogs,organic or inorganic compounds (i.e., including heteroorganic andorganometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

The term “antibiotics” is used herein to describe a compound orcomposition which decreases the viability of a microorganism, or whichinhibits the growth or reproduction of a microorganism. As used in thisdisclosure, an antibiotic is further intended to include anantimicrobial, bacteriostatic, or bactericidal agent. Exemplaryantibiotics include, but are not limited to, penicillins,cephalosporins, penems, carbapenems, monobactams, aminoglycosides,sulfonamides, macrolides, tetracyclins, lincosides, quinolones,chloramphenicol, vancomycin, metronidazole, rifampin, isoniazid,spectinomycin, trimethoprim, and sulfamethoxazole.

The term “therapeutic agents” is art-recognized and refers to anychemical moiety that is a biologically, physiologically, orpharmacologically active substance that acts locally or systemically ina subject. Examples of therapeutic agents, also referred to as “drugs”,are described in well-known literature references such as the MerckIndex, the Physicians Desk Reference, and The Pharmacological Basis ofTherapeutics, and they include, without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of a disease or illness;substances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a physiological environment. Various forms of atherapeutic agent may be used which are capable of being released fromthe subject composition into adjacent tissues or fluids uponadministration to a subject. Examples include steroids and esters ofsteroids (e.g., estrogen, progesterone, testosterone, androsterone,cholesterol, norethindrone, digoxigenin, cholic acid, deoxycholic acid,and chenodeoxycholic acid), boron-containing compounds (e.g.,carborane), chemotherapeutic nucleotides, drugs (e.g., antibiotics,antivirals, antifungals), enediynes (e.g., calicheamicins, esperamicins,dynemicin, neocarzinostatin chromophore, and kedarcidin chromophore),heavy metal complexes (e.g., cisplatin), hormone antagonists (e.g.,tamoxifen), non-specific (non-antibody) proteins (e.g., sugaroligomers), oligonucleotides (e.g., antisense oligonucleotides that bindto a target nucleic acid sequence (e.g., mRNA sequence)), peptides,proteins, antibodies, photodynamic agents (e.g., rhodamine 123),radionuclides (e.g., I-131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89,Ho-166, Sm-153, Cu-67 and Cu-64), toxins (e.g., ricin), andtranscription-based pharmaceuticals.

As used herein, the term “hormones” generally refers to naturally ornon-naturally occurring hormones, analogues and mimics thereof. Incertain embodiments, the term “hormones” refers to any hormones used intherapeutic treatment, e.g., growth hormone treatment. As used herein,“growth hormone” or “GH” refers to growth hormone in native-sequence orin variant form, and from any source, whether natural, synthetic, orrecombinant. Examples include human growth hormone (hGH), which isnatural or recombinant GH with the human native sequence (somatotropinor somatropin), and recombinant growth hormone (rGH), which refers toany GH or variant produced by means of recombinant DNA technology,including somatrem, somatotropin, and somatropin. In one embodiment,hormones include insulin.

As used herein, a “contrast agent” can be any chemical moiety that isused to increase the degree of difference between the lightest anddarkest part of a scan or an imaging, e.g., during medical scan orimaging, relative to a scan performed without the use of a contrastagent. For example, contrast agents can include imaging agentscontaining radioisotopes such as indium or technetium; dyes containingiodine, gadolinium or cyanine; enzymes such as horse radish peroxidase,GFP, alkaline phosphatase, or β-galactosidase; fluorescent substancessuch as europium derivatives; luminescent substances such asN-methylacrydium derivatives or the like. In some embodiments, contrastagents can include gold nanoparticles and/or quantum dots.

As used herein, the term “substantially” means a proportion of at leastabout 60%, or preferably at least about 70% or at least about 80%, or atleast about 90%, at least about 95%, at least about 97% or at leastabout 99% or more, or any integer between 70% and 100%. In someembodiments, the term “substantially” means a proportion of at leastabout 90%, at least about 95%, at least about 98%, at least about 99% ormore, or any integer between 90% and 100%. In some embodiments, the term“substantially” can include 100%.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to the components thereof as describedherein, which are exclusive of any element not recited in thatdescription of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in diseases and disorders, separation and detection techniques canbe found in The Merck Manual of Diagnosis and Therapy, 18th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); andRobert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8).

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

All patents and other publications identified throughout thespecification are expressly incorporated herein by reference for thepurpose of describing and disclosing, for example, the methodologiesdescribed in such publications that might be used in connection with thepresent invention. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents are based on theinformation available to the applicants and do not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

Some embodiments described herein are further illustrated by thefollowing example which should not be construed as limiting.

The contents of all references cited throughout this application,examples, as well as the figures and tables are incorporated herein byreference in their entirety.

EXAMPLES Example 1 Fabrication of Exemplary Injectable SilkFibroin-Based Foams

A silk fibroin foam sheet can be produced by any art-recognized methods.In this Example, a silk foam sheet was created by using afreezer-processing technique, for example, freezer-processing of a silkfibroin solution directly.

To prepare a silk fibroin solution, Bombyx mori silkworm cocoonspurchased from Tajimia Shoji Co. (Yokohama, Japan) or a Taiwanesesupplier were cut into pieces, and boiled in 0.02 M Na₂CO₃ for about10-60 minutes, and preferably for about 30 minutes. The resulting silkfibroin fibers were rinsed in distilled water and let dried. The driedsilk fibroin fibers were re-solubilized in 9.3 M LiBr at 60° C., forabout 1-4 hours, until dissolved. The silk fibroin solution wasdialyzed, with a molecular weight cutoff of 3500 Daltons, againstdistilled water for at least 6 water changes.

The silk fibroin solution (e.g., with a concentration of about 1% toabout 6% w/v), made from Japanese cocoons or Taiwanese cocoons, waspoured into a container (e.g., a plastic Petri dish). The silk fibroinsolution was then stored and maintained at around 20° F. (˜−7° C.) forabout 3 days (e.g., stored in an EdgeStar Model FP430 thermoelectriccooler). The resultant silk fibroin material was gel-like, but not astiff solid. The gel-like silk fibroin material was then freeze-driedfor about 3 days (e.g., using a VirTis Genesis (Model 25L Genesis SQSuper XL-70) Lyophilizer). After removal from the lyophilizer, thefreeze-dried silk fibroin material became a foam-like material with avery consistent interconnected fine-pore structure. In some embodiments,the silk fibroin foam was further soaked in alcohol (e.g., about 70%methanol) to induce beta sheet formation. In such embodiments, there canbe about 10% shrinkage in volume of the silk fibroin foam sheet aftertreatment with alcohol. The alcohol-treated foam sheet exhibitedexcellent stiffness and toughness. A 4-mm diameter biopsy punch (FIG.1A) was then used to cut out small silk fibroin foam disks(approximately 2 mm thickness) from the silk fibroin foam sheet, asshown in FIG. 1B.

Example 2 Evaluation of the Silk Fibroin-Based Foams for Injection intoa Tissue

To assess the potential for injecting one or more embodiments of theinjectable silk fibroin-based foam constructs into a tissue, anexperiment was conducted on raw chicken thighs. FIG. 2A shows a silkfibroin-based foam loaded into a sharpened conical-shaped applicatortip, e.g., a pipette tip. The conical interior space of a pipette tipcan allow the foam to be ejected with less friction and force than astraight applicator tip (e.g., a straight needle). The foam was pushedthrough the pipette tip using a stiff wire (FIG. 2B). By way of exampleonly, in some embodiments, injection of the silk fibroin-based foam intoa raw chicken thigh tissue was performed as described below. A straight14-gauge needle (˜1.6 mm inner diameter) was first used to puncture ahole in the meaty part of the chicken thigh (FIG. 3A). The pipette tiploaded with the silk fibroin-based foam was inserted into the hole (FIG.3B) and slowly drawn out, while the stiff wire was used to eject thefoam (FIG. 3C). FIG. 3D shows the foam injected and positioned in theraw chicken thigh. Thus, the silk fibroin-based foams can be injectedinto a tissue, e.g., for filling a void in the tissue or augmenting thetissue.

FIGS. 4A-4F show the injected silk fibroin-based foam being excised. Forexample, a razor blade was used to slice through the raw chicken meat(FIGS. 4A through 4D). Using tweezers, the silk fibroin-based foam wasthen extracted (FIGS. 4E and 4F). FIG. 4F shows that the extracted silkfibroin-based foam was intact, indicating that the silk fibroin-basedfoam can remain intact after injection into a tissue.

Example 3 In Vivo Studies of Injectable Silk Fibroin-Based Foams

A rat or mouse model was used for assessing some embodiments of the silkfibroin-based foams described herein. Other mammalian models (e.g.,rabbit, canine, or porcine models) can also be used depending on theapplications of the injectable silk fibroin-based foams and the tissuesto be modeled for treatment. The rats or mice were weighed andanesthetized with isoflurane in oxygen prior to injection. A silkfibroin-based foam having a size of about 5 mm in diameter by about 2 mmin height was used for injection. Briefly, dry silk fibroin foams wereimmersed in saline immediately before loading into a catheter.Alternatively, the dry silk fibroin foam could be immersed inlipoaspirate immediately before loading into a catheter (e.g., as shownin FIG. 5). Subcutaneous injections were performed above the pectoralmuscles. Intramuscular and submuscular injections were performed betweenthe pectoralis major and pectoralis minor muscles or underneath thepectoral muscles, respectively. A fanning subcutaneous injection methodwas performed in the dorsus of the rat or the mouse. Injected sampleswere explanted, and evaluated for volume retention after 1, 14, 40 and60 days. Volume retention was performed by 2 methods, e.g., scalemeasurements and volume displacement.

A rod-and-plunger system can be used to inject a silk fibroin foam intoa tissue. For example, FIG. 7A shows an exemplary Hauptner syringe thatwas custom-modified to inject a silk fibroin foam subcutaneously invivo, e.g., in a mouse or rat model. The design is, at least in part,based on a commercially available pistol-style (Hauptner) syringe madeby Ideal Instruments. This Hauptner syringe device has a spring-loadedhandle that generally forces an Injector Drawrod into the syringe bodyby a pre-set distance (using the Injection Stroke Adjuster). To modifythe Hauptner syringe device for injecting a foam, rather than a solutionor a gel, a Foam Ramrod was manufactured to fit through the end of thesyringe, where the Catheter Adaptor is located, as shown in FIG. 7A. Acatheter is attached to the adaptor. Typically, a catheter is a tubethat is used to remove fluid from the body. In some embodiments, atapered catheter (i.e., the barrel of the catheter is larger than thecatheter tip) can be used to inject a silk fibroin foam into a tissue.The taper allows a foam to be pre-positioned in the barrel beforeattaching the catheter to the Hauptner syringe and allows the foam to begradually compressed during the process of injection.

By way of example only, the rat or mouse study protocol involved theinitial creation of a small hole in the rat or mouse skin using a 14gauge needle positioned within the catheter (e.g., as shown in FIG. 7B,step 1: left panel). The outer diameter of the catheter was small enoughto allow penetration into the hole, while the inner diameter was largeenough to allow passage of the compressed foam into the subcutaneousarea of the rat or the mouse. FIGS. 7A-7B shows the exemplary stages ofinjecting a silk fibroin foam in an animal study. The Foam Ramrod isinserted into the syringe (FIG. 7A). The needle placed within thecatheter is used to facilitate the insertion of the catheter in thedesired position (FIG. 7B, step 1). After placing the catheter in thedesired position within a tissue, the needle can be removed from theneedle/catheter. The silk fibroin foam is positioned in the barrel ofthe catheter using tweezers (FIG. 7B, step 2). The catheter loaded withthe silk fibroin foam is then connected to the Catheter Adapter of theinjection gun (FIG. 7B, step 3). The Injection Handle of the injectiongun is then repeatedly squeezed to slowly inject the foam into theanimal (FIG. 7B, step 4).

FIGS. 8A-8C show images of silk fibroin-based foams injected into a ratmodel in vivo after the removal of the rat skin. Silk fibroin-basedfoams produced from different concentrations of silk fibroin solution(e.g., 1%, 3%, 6% silk fibroin) and sources of cocoon (Japanese: JP vs.Taiwanese: TW) were evaluated after injection for 1 day (FIG. 8A), 14days (FIG. 8B) and 30 days (FIG. 8C). FIG. 8A shows that the injectedsilk fibroin-based foams remained clear 1 day after injection, unlessthey were stained by blood due to a puncture into a blood vessel (e.g.,TW3). FIGS. 8B-8C show that the injected silk fibroin-based foamsobtained a reddish hue about 14 days and about 30 days, respectively,after injection. However, there appeared no significant change invascularization leading to the injected foams. FIG. 8D shows an image ofthe injected foams visible from outside skin of a rat. FIG. 8E is a setof images showing gross morphology of the silk fibroin-based injectablefoams (corresponding to the ones in FIGS. 8A-8C) explanted after anindicated post-injection period (e.g., 1 day, 14 days and 30 dayspost-injection). There are no observable visual differences in grossmorphology at the indicated timepoints. The silk fibroin foams areconsistently stiffer with increased silk weight percentage. All explantsare soft to the touch and return to their original shape afterdeformation. Histology for the explanted silk fibroin foams attached tothe tissue is performed to evaluate vascularity and integration withtissues.

FIG. 8F shows the volume retention results of the silk fibroin-basedfoams after injection into the rat tissue for 1 day or 14 days. FIG. 8Gshows the volume retention results of the silk fibroin-based foams afterinjection into the tissue for 14 days, 30 days or 60 days. The resultsof FIGS. 8F and 8G are expressed in percents of volume retained relativeto the original volume (i.e., the volume of the silk fibroin-based foamsbefore compression). FIGS. 8F and 8G show that the silk fibroin foamsproduced from a silk fibroin solution of about 1%, 3%, or 6% canmaintain at least about 80% of their original volume (including at leastabout 90%, at least about 95%, at least about 100% or higher, of theiroriginal volume) for at least about 30 days or longer, and at leastabout 50% or higher of their original volume for at least about 60 daysor longer. As shown in FIGS. 8F-8G, the volume retained in the tissuecan be greater than the original volume, likely because the silkfibroin-based foams can absorb water and thus swell. The stiffness ofthe silk fibroin foams generally increases with the concentration of thesilk fibroin solution. Thus, silk fibroin foams of higher silk fibroinconcentrations can generally maintain their volume for a longer periodof time than those of lower silk fibroin concentrations. Further, thesilk fibroin foams produced from a silk fibroin solution of about 1%, 3%or 6% remain soft and spongy for at least 60 days after injection intothe rat.

Presented herein are some embodiments of the silk fibroin-based foamsthat can be ejected from a needle, pipette tip, catheter or othertubular structures (e.g., including tubular structures with a taperedend). The silk fibroin-based foams can be compressed prior to injectionand then expand, for example, by at least about 2.5-fold, upon releasedfrom the compression and/or upon injection into a tissue. In someembodiments, the injected silk fibroin-based foam can have sufficientphysical and mechanical integrity to bulge the surface of raw chickenmeat and provide a noticeable bulk when injected subcutaneously in a rat(FIG. 8D). The properties of the silk fibroin-based foam can becontrolled by various factors, including, but not limited to, degummingtime during silk fibroin solution preparation, the concentration of silkfibroin solution used, and the use of a methanol or other suitabletreatment to control crystalline (beta sheet) content.

In some embodiments, longer silk fibroin-based foam constructs can beused to fill relatively large soft tissue void spaces through anincision, cannula, needle, pipette tip, catheter, or other tubularstructures (e.g., including tubular structures with a tapered end), thusrequiring a relatively small hole to be used for tissue penetration, ascompared to conventional invasive procedures.

What is claimed is:
 1. A method for repairing or augmenting a tissue ina subject comprising: placing into the tissue to be repaired oraugmented a composition comprising a compressed silk fibroin matrix,wherein the compressed silk fibroin matrix expands upon placement intothe tissue, and retains at least about 1% of its original expandedvolume within the tissue for at least about 2 weeks, wherein the silkfibroin matrix has a porosity of at least 50%.
 2. The method of claim 1,wherein the compressed silk fibroin matrix expands in volume by at leastabout 2-fold, relative to the volume of the compressed silk fibroinmatrix.
 3. The method of claim 1, wherein the silk fibroin matrixretains at least about 50% of its original expanded volume within thetissue for at least about 2 weeks, at least about 6 weeks, at leastabout 3 months, or at least about 6 months.
 4. The method of claim 1,wherein the pores have a size of about 1 μm to about 1500 μm.
 5. Themethod of claim 1, wherein the silk fibroin matrix is formed from a silkfibroin solution of about 0.1% w/v to about 30% w/v.
 6. The method ofclaim 1, wherein the composition or the silk fibroin matrix furthercomprises at least one active agent.
 7. The method of claim 1, whereinthe composition further comprises a cell.
 8. The method of claim 1,wherein the composition further comprises a biological fluid orconcentrate.
 9. The method of claim 1, wherein the composition or thesilk fibroin matrix further comprises a hydrogel.
 10. The method ofclaim 1, wherein the composition further comprises a carrier.
 11. Themethod of claim 1, wherein the tissue is a soft tissue.
 12. Aninjectable composition for use in repairing or augmenting a tissue in asubject, comprising a compressed silk fibroin matrix, wherein thecompressed silk fibroin matrix expands upon injection into the tissue,and retains at least about 1% of its original expanded volume within thetissue for at least about 2 weeks, wherein the silk fibroin matrix has aporosity of at least 50%.
 13. The composition of claim 12, wherein thecompressed silk fibroin matrix expands in volume by at least about2-fold, relative to the volume of the compressed silk fibroin matrix.14. The composition of claim 12, wherein the silk fibroin matrix retainsat least about 50% of its original expanded volume within the tissue forat least about 2 weeks, at least about 6 weeks, at least about 3 months,or at least about 6 months.
 15. The composition of claim 12, wherein thepores have a size of about 1 μm to about 1500 μm.
 16. The composition ofclaim 12, wherein the silk fibroin matrix is formed from a silk fibroinsolution of about 0.1% w/v to about 30% w/v.
 17. The composition ofclaim 12, wherein the injectable composition or the silk fibroin matrixfurther comprises at least one active agent.
 18. The composition ofclaim 12, further comprising a cell.
 19. The composition of claim 12,further comprising a biological fluid or concentrate.
 20. Thecomposition of claim 12, wherein the injectable composition or the silkfibroin matrix further comprises a hydrogel.
 21. The composition ofclaim 12, wherein the injectable composition further comprises acarrier.
 22. The composition of claim 12, wherein the compressed silkfibroin matrix has a volume of about 10% to about 90% of its originalvolume before compression.
 23. A delivery device comprising aninjectable composition of claim
 12. 24. The composition of claim 21,wherein the carrier is or comprises lipoaspirate.
 25. The composition ofclaim 18, wherein the cell is or comprises at least one of a stem cell,adipose cell, or adipose-derived stem cell.